Dual Targeting siRNA  Agents

ABSTRACT

The invention relates to dual targeting siRNA agents targeting a PCSK9 gene and a second gene, and methods of using dual targeting siRNA agents to inhibit expression of PCSK9 and to treat PCSK9 related disorders, e.g., hyperlipidemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/497,226, filed Oct. 10, 2012, (allowed), which is National Stage of International Application No. PCT/US2010/049868, filed Sep. 22, 2010, which claims the benefit of U.S. Provisional Application No. 61/244,859, filed Sep. 22, 2009, and claims the benefit of U.S. Provisional Application No. 61/313,584, filed Mar. 12, 2010, all of which are hereby incorporated in their entirety by reference.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically as a text file named 30503_US_sequencelisting.txt, created on Oct. 16, 2015, with a size of 1,351,680 bytes. The sequence listing is incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a composition of two covalently linked siRNAs, e.g., a dual targeting siRNA agent. At least one siRNA is a dsRNA that targets a PCSK9 gene. The covalently linked siRNA agent is used in methods of inhibition of PCSK9 gene expression and methods of treatment of pathological conditions associated with PCSK9 gene expression, e.g., hyperlipidemia.

BACKGROUND OF THE INVENTION

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family. The other eight mammalian subtilisin proteases, PCSK1-PCSK8 (also called PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, and S1P/SKI-1) are proprotein convertases that process a wide variety of proteins in the secretory pathway and play roles in diverse biological processes (Bergeron, F. (2000) J. Mol. Endocrinol. 24, 1-22, Gensberg, K., (1998) Semin. Cell Dev. Biol. 9, 11-17, Seidah, N. G. (1999) Brain Res. 848, 45-62, Taylor, N. A., (2003) FASEB J. 17, 1215-1227, and Zhou, A., (1999) J. Biol. Chem. 274, 20745-20748). PCSK9 has been proposed to play a role in cholesterol metabolism. PCSK9 mRNA expression is down-regulated by dietary cholesterol feeding in mice (Maxwell, K. N., (2003) J. Lipid Res. 44, 2109-2119), up-regulated by statins in HepG2 cells (Dubuc, G., (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1454-1459), and up-regulated in sterol regulatory element binding protein (SREBP) transgenic mice (Horton, J. D., (2003) Proc. Natl. Acad. Sci. USA 100, 12027-12032), similar to the cholesterol biosynthetic enzymes and the low-density lipoprotein receptor (LDLR). Furthermore, PCSK9 missense mutations have been found to be associated with a form of autosomal dominant hypercholesterolemia (Hchola3) (Abifadel, M., et al. (2003) Nat. Genet. 34, 154-156, Timms, K. M., (2004) Hum. Genet. 114, 349-353, Leren, T. P. (2004) Clin. Genet. 65, 419-422). PCSK9 may also play a role in determining LDL cholesterol levels in the general population, because single-nucleotide polymorphisms (SNPs) have been associated with cholesterol levels in a Japanese population (Shioji, K., (2004) J. Hum. Genet. 49, 109-114).

Autosomal dominant hypercholesterolemias (ADHs) are monogenic diseases in which patients exhibit elevated total and LDL cholesterol levels, tendon xanthomas, and premature atherosclerosis (Rader, D. J., (2003) J. Clin. Invest. 111, 1795-1803). The pathogenesis of ADHs and a recessive form, autosomal recessive hypercholesterolemia (ARH) (Cohen, J. C., (2003) Curr. Opin. Lipidol. 14, 121-127), is due to defects in LDL uptake by the liver. ADH may be caused by LDLR mutations, which prevent LDL uptake, or by mutations in the protein on LDL, apolipoprotein B, which binds to the LDLR. ARH is caused by mutations in the ARH protein that are necessary for endocytosis of the LDLR-LDL complex via its interaction with clathrin. Therefore, if PCSK9 mutations are causative in Hchola3 families, it seems likely that PCSK9 plays a role in receptor-mediated LDL uptake.

Overexpression studies point to a role for PCSK9 in controlling LDLR levels and, hence, LDL uptake by the liver (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) J. Biol. Chem. 279, 50630-50638). Adenoviral-mediated overexpression of mouse or human PCSK9 for 3 or 4 days in mice results in elevated total and LDL cholesterol levels; this effect is not seen in LDLR knockout animals (Maxwell, K. N. (2004) Proc. Natl. Acad. Sci. USA 101, 7100-7105, Benjannet, S., et al. (2004) J. Biol. Chem. 279, 48865-48875, Park, S. W., (2004) 1 Biol. Chem. 279, 50630-50638). In addition, PCSK9 overexpression results in a severe reduction in hepatic LDLR protein, without affecting LDLR mRNA levels, SREBP protein levels, or SREBP protein nuclear to cytoplasmic ratio.

Loss of function mutations in PCSK9 have been designed in mouse models (Rashid et al., (2005) PNAS, 102, 5374-5379), and identified in human individuals (Cohen et al. (2005) Nature Genetics 37:161-165). In both cases loss of PCSK9 function lead to lowering of total and LDLc cholesterol. In a retrospective outcome study over 15 years, loss of one copy of PCSK9 was shown to shift LDLc levels lower and to lead to an increased risk-benefit protection from developing cardiovascular heart disease (Cohen et al., (2006) N. Engl. J. Med., 354:1264-1272).

X-box binding protein 1 (XBP-1) is a basic leucine zipper transcription factor that is involved in the cellular unfolded protein response (UPR). XBP-1 is known to be active in the endoplasmic reticulum (ER). The ER consists of a system of folded membranes and tubules in the cytoplasm of cells. Proteins and lipids are manufactured and processed in the ER. When unusual demands are placed on the ER, “ER stress” occurs. ER stress can be triggered by a viral infection, gene mutations, exposure to toxins, aggregation of improperly folded proteins or a shortage of intracellular nutrients. The result can be Type II diabetes, metabolic syndrome, a neurological disorder or cancer.

Two XBP-1 isoforms are known to exist in cells: spliced XBP-1S and unspliced XBP-1U. Both isoforms of XBP-1 bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. HTLV-1 is associated with a rare form of blood dyscrasia known as Adult T-cell Leukemia/lymphoma (ATLL) and a myelopathy, tropical spastic paresis.

Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.

A description of siRNA targeting PCSK9 can be found in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487).

A description of siRNA targeting XPB-1 can be found in U.S. patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and published as US 2009-0275638.

Dual targeting siRNAs can be found in International patent application publication no. WO/2007/091269.

SUMMARY OF THE INVENTION

Described herein are dual targeting siRNA agent in which a first siRNA targeting PCSK9 is covalently joined to a second siRNA targeting a gene implicated in cholesterol metabolism, e.g., XBP-1. The two siRNAs are covalently linked via, e.g., a disulfide linker.

Accordingly one aspect of the invention is a dual targeting siRNA agent having a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein the first dsRNA and the second dsRNA are linked with a covalent linker. The second gene is can be, e.g., XBP-1, PCSK9, PCSK5, ApoC3, SCAP, or MIG12. In one embodiment, the second gene is XBP-1. Each dsRNA is 30 nucleotides or less in length. In general, each strand of each dsRNA is 19-23 bases in length.

In one embodiment, the dual targeting siRNA agent comprising a first dsRNA AD-10792 targeting a PCSK9 gene and a second dsRNA AD-18038 targeting an XBP-1 gene, wherein AD-10792 sense strand and AD-18038 sense strand are covalently linked with a disulfide linker.

The first dsRNA of the dual targeting siRNA agent targets a PCSK9 gene. In one aspect, the first dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or includes an antisense strand of one of Tables 1, 2, or 4-8, or includes a sense strand and an antisense strand of one of Tables 1, 2, or 4-8. The first dsRNA can be AD-9680 or AD-10792.

In some embodiments, the second dsRNA target XBP-1. In one aspect, the second dsRNA includes at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or includes an antisense strand of one of Tables 3 or 9-13, or includes a sense strand and an antisense strand of one of Tables 3 or 9-13. For example, the second dsRNA can be AD-18038.

Either the first and second dsRNA can include at least one modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In some embodiments, the first and second dsRNAs include “endo-light” modification with 2′-O-methyl modified nucleotides and nucleotides comprising a 5′-phosphorothioate group.

The first and second dsRNAs are linked with a covalent linker. In some embodiments, the linker is a disulfide linker. Various combinations of strands can be linked, e.g., the first and second dsRNA sense strands are covalently linked or, e.g., the first and second dsRNA antisense strands are covalently linked. In some embodiments, any of the dual targeting siRNA agents of the invention include a ligand.

Also included in the invention are isolated cells having and vectors encoding the dual targeting siRNA agent described herein.

In one aspect, administration of the dual targeting siRNA agent to a cell inhibits expression of the PCSK9 gene and the second gene at a level equivalent to inhibition of expression of both genes using administration of each siRNA individually. In another aspect, administration of the dual targeting siRNA agent to a subject results in a greater reduction of total serum cholesterol that that obtained by administration of each siRNA alone.

The invention also includes a pharmaceutical composition comprising the dual targeting siRNA agents described herein and a pharmaceutical carrier. In one embodiment, the pharmaceutical carrier is a lipid formulation, e.g., a lipid formulation including cationic lipid DLinDMA or cationic lipid XTC. Examples of lipid formulations described in (but not limited to) Table A, below. The lipid formulation can be XTC/DSPC/Cholesterol/PEG-DMG at % mol ratios of 50/10/38.5/1.5.

Another aspect of the invention includes methods of using the dual targeting siRNA agents described herein. In one embodiment, the invention is a method of inhibiting expression of the PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the any of the dual targeting siRNA agents and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.

In another embodiment, the invention includes methods of treating a disorder mediated by PCSK9 expression with the step of administering to a subject in need of such treatment a therapeutically effective amount of the pharmaceutical compositions described herein. In one aspect, the disorder is hyperlipidemia. In still another embodiment, the invention includes methods of reducing total serum cholesterol in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical compositions described herein.

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the effect on PCSK9 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent only FIG. 1B is a graph showing the effect on XBP-1 mRNA levels in primary mouse hepatocytes following treatment with a dual targeting siRNA, AD-23426. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA. Lipo2000: control transfection agent only.

FIG. 2 is a graph showing the effect on PCSK9 and XBP-1 mRNA levels in mice following treatment with a dual targeting siRNA, AD-23426. LNP09 (lipid) formulated siRNA was administered to mice as described. AD-23426 was as effective at reducing mRNA expression as each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.

FIG. 3 is a graph showing the effect on serum cholesterol levels in mice following treatment with a dual targeting siRNA, AD-23426. LNP09 (lipid) formulated siRNA was administered to mice as described. AD-23426 was more effective at reducing serum cholesterol compared to each single gene target siRNA. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.

FIG. 4A is a graph showing the effect on IFN-α in human PBMC following treatment with a dual targeting siRNA, AD-23426. FIG. 4B is a graph showing the effect on TNF-α in human PBMC following treatment with a dual targeting siRNA, AD-23426. DOTAP and LNP09 (lipid) formulated siRNAs was administered huPBMC as described below. AD-23426 did not induce IFN-α or TNF-α.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a solution to the problem of treating diseases that can be modulated by the down regulation of the PCSK9 gene, such as hyperlipidemia, by using dual targeting siRNA to silence the PCSK9 gene.

The invention provides compositions and methods for inhibiting the expression of the PCSK9 gene in a subject using two siRNA, e.g., a dual targeting siRNA. The invention also provides compositions and methods for treating pathological conditions and diseases, such as hyperlipidemia, that can be modulated by down regulating the expression of the PCSK9 gene.

The dual targeting siRNA agents target a PCSK9 gene and at least one other gene. The other gene can be another region of the PCSK9 gene, or can be another gene, e.g., XBP-1.

The dual targeting siRNA agents have the advantage of lower toxicity, lower off-target effects, and lower effective concentration compared to individual siRNAs.

The use of the dual targeting siRNA dsRNAs enables the targeted degradation of an mRNA that is involved in the regulation of the LDL receptor and circulating cholesterol levels. Using cell-based and animal assays it was demonstrated that inhibiting both a PCSK9 gene and an XBP-1 gene using a dual targeting siRNA is at least as effective at inhibiting their corresponding targets as the use of single siRNAs. It was also demonstrated that administration of a dual targeting siRNA results in a synergistic lowering of total serum cholesterol. Thus, reduction of total serum cholesterol is enhanced with a dual targeting siRNA compared to a single target siRNA.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The term “PCSK9” refers to the proprotein convertase subtilisin kexin 9 gene or protein (also known as FH3, HCHOLA3, NARC-1, NARC1). Examples of mRNA sequences to PCSK9 include but are not limited to the following: human: NM_174936; mouse: NM_153565, and rat: NM_199253. Additional examples of PCSK9 mRNA sequences are readily available using, e.g., GenBank.

The term “XBP-1” refers to -Box Protein 1, which is also known as Tax-responsive element-binding protein 5, TREB5, and XBP2. XBP-1 sequence can be found as NCBI GeneID:7494 and RefSeq ID number:NM_005080 (human) and NM_013842 (mouse). A dsRNA featured in the invention can target a specific XBP-1 isoform, e.g., the spliced form (XBP-1S) or the unspliced form (XBP-1U), or a dsRNA featured in the invention can target both isoforms by binding to a common region of the mRNA transcript.

The term “PCSK5” refers to the Proprotein convertase subtilisin/kexin type 5 gene, mRNA or protein belonging to the subtilisin-like proprotein convertase family.

The term “ApoC3” refers to the Apolipoprotein C-III protein gene, mRNA or protein, and is a very low density lipoprotein (VLDL).

The term “SCAP” refers to the SREBP cleavage-activating protein gene, mRNA or protein. SCAP is a regulatory protein that is required for the proteolytic cleavage of the sterol regulatory element binding protein (SREBP). Example of siRNA targeting SCAP are described in U.S. patent application Ser. No. 11/857,120, filed on Sep. 18, 2007, published as US 20090093426. This application and the siRNA sequences described therein are incorporated by reference for all purposes.

The term “MIG12” is a gene also known as TMSB10 and TB10 refers to the thymosin beta 10 gene. Example of siRNA targeting MIG12 are described International patent application no. PCT/US10/25444, filed on Feb. 25, 2010, published as WO/20XX/XXXXXX. This application and the siRNA sequences described therein are incorporated by reference for all purposes.

As used herein, the term “iRNA” refers to an agent that contains RNA and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. The term iRNA includes siRNA.

As described in more detail below, the term “siRNA” and “siRNA agent” refers to a dsRNA that mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.

A “double-stranded RNA” or “dsRNA,” as used herein, refers to an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA.

The term “dual targeting siRNA agent” refers to a composition of two siRNAs, e.g., two dsRNAs. One dsRNA includes an antisense strand with a first region of complementarity to a first target gene, e.g., PCSK9. The second dsRNA include an antisense strand with a second region of complementarity to a second target gene. In some embodiments, the first and second target genes are identical, e.g., both are PCSK9 and each dsRNA targets a different region of PCSK9. In other embodiments, the first and second target genes are different, e.g., the first dsRNA targets PCSK9 and the second dsRNA targets a different gene, e.g., XBP-1.

“Covalent linker” refers to a molecule for covalently joining two molecules, e.g., two dsRNAs. As described in more detail below, the term includes, e.g., a nucleic acid linker, a peptide linker, and the like and includes disulfide linkers.

The term “target gene” refers to a gene of interest, e.g., PCSK9 or a second gene, e.g., XBP-1, targeted by an siRNA of the invention for inhibition of expression.

As described in more detail below, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of the target gene (e.g., an mRNA encoding PCSK9 or a second gene, e.g., XBP-1). For example, a polynucleotide is complementary to at least a part of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding PCSK9.

The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleotide, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term “iRNA.”

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA. One or more of the nucleotides in the overhang can be replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.

“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.

As used herein, the term “modulate the expression of,” refers to at an least partial “inhibition” or partial “activation” of target gene expression in a cell treated with an iRNA composition as described herein compared to the expression of the target gene in an untreated cell.

The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to a target gene, herein refer to the at least partial activation of the expression of a target gene, as manifested by an increase in the amount of target mRNA, which may be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of a target gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

In one embodiment, expression of a target gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, a target gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention. In some embodiments, expression of a target gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the target gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848, each of which is incorporated herein by reference.

The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of,” and the like, in so far as they refer to a target gene, herein refer to the at least partial suppression of the expression of a target gene, as manifested by a reduction of the amount of target mRNA which may be isolated from or detected in a first cell or group of cells in which a target gene is transcribed and which has or have been treated such that the expression of target gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production. In principle, target gene silencing may be determined in any cell expressing target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of the target gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of a target gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA featured in the invention. In some embodiments, a target gene is suppressed by at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by administration of an iRNA as described herein.

As used herein in the context of target gene expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by target expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by target expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition.

By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.

As used herein, the phrase “therapeutically effective amount” refers to an amount that provides a therapeutic benefit in the treatment or management of pathological processes mediated by target gene expression, e.g., PCSK9 and/or a second gene, e.g., XBP-1, or an overt symptom of pathological processes mediated target gene expression. The phrase “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the prevention of pathological processes mediated by target gene expression or an overt symptom of pathological processes mediated by target gene expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by target gene expression, the patient's history and age, the stage of pathological processes mediated by target gene expression, and the administration of other agents that inhibit pathological processes mediated by target gene expression.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological or therapeutic result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter.

The term “pharmaceutically carrier” refers to a carrier for administration of a therapeutic agent, e.g., a dual targeting siRNA agent. Carriers are described in more detail below, and include lipid formulations, e.g., LNP09 and SNALP formulations.

Double-Stranded Ribonucleic Acid (dsRNA)

Described herein are dual targeting siRNA agents, e.g., siRNAs that inhibit the expression of a PCSK9 gene and a second gene. The dual targeting siRNA agent includes two siRNA covalently linked via, e.g., a disulfide linker. The first siRNA targets a first region of a PCSK9 gene. The second siRNA targets a second gene, e.g., XBP-1, or, e.g., targets a second region of the PCSK9 gene.

The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Applied Biosystems, Inc. Further descriptions of synthesis are found below and in the examples.

Each siRNA is a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a target gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.

Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.”

Generally, the duplex structure of the siRNA, e.g., dsRNA, is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs.

The two siRNAs in the dual targeting siRNA agent can have duplex lengths that are identical or that differ.

The region of complementarity to the target sequence in an siRNA is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. The region of complementarity can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides. In some embodiments the target sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

The two siRNAs in the dual targeting siRNA agent can have regions of complementarity that are identical in length or that differ in length.

Any of the dsRNA, e.g., siRNA as described herein may include one or more single-stranded nucleotide overhangs. In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, or 1 or 2 or 3 or 4 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. The two siRNAs in the dual targeting siRNA agent can have different or identical overhangs as described by location, length, and nucleotide.

The dual targeting siRNA agent includes at least a first siRNA targeting a first region of a PCSK9 gene. In one embodiment, a PCSK9 gene is a human PCSK9 gene. In another embodiment the PCSK9 gene is a mouse or a rat PCSK9 gene. Exemplary siRNA targeting PCSK9 are described in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161). Additional disclosure can be found in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.

Tables 1, 2, and 4-8 disclose sequences of the target, sense strands, and antisense strands of PCSK9 targeting siRNA.

In one embodiment the first siRNA is AD-9680. The dsRNA AD-9680 targets the human PCSK 9 gene at nucleotides 3530-3548 of a human PCSK9 gene (accession number NM_174936).

TABLE 1 AD-9680 siRNA sequences SEQ ID Table 1: AD-9680 Sequence 5′ to 3′ NO: Target sequence UUCUAGACCUGUUUUGCUU 4142 Sense strand UUCUAGACCUGUUUUGCUU 4143 Sense strand, uucuAGAccuGuuuuGcuuTsT  4144 modified Antisense strand AAGCAAAACAGGUCUAGAA 4145 Antisense strand, AAGcAAAAcAGGUCuAGAATsT  4146 modified

In another embodiment, the first siRNA is AD-10792. The dsRNA AD-10792 targets the PCSK9 gene at nucleotides 1091-1109 of a human PCSK9 gene (accession number NM_174936). AD-10792 is also complementary to rodent PCSK9.

TABLE 2 AD-10792 siRNA sequences SEQ ID Table 2: AD-10792 Sequence 5′ to 3′ NO: Target sequence GCCUGGAGUUUAUUCGGAA 4147 Sense strand GCCUGGAGUUUAUUCGGAA 4148 Sense strand, GccuGGAGuuuAuucGGAATsT  4149 modified Antisense strand UUCCGAAUAAACUCCAGGC 4150 Antisense strand, UUCCGAAuAAACUCcAGGCTsT  4151 modified

The second siRNA of the dual targeting siRNA agent targets a second gene. In one embodiment, the second gene is PCSK9, and the second siRNA target a region of PCSK9 that is different from the region targeted by the first siRNA.

Alternatively, the second siRNA targets a different second gene. Examples include genes that interact with PCSK9 and/or are involved with lipid metabolism or cholesterol metabolism. For example, the second target gene can be XBP-1, PCSK5, ApoC3, SCAP, MIG12, HMG CoA Reductase, or IDOL (Inducible Degrader of the LDLR) and the like. In one embodiment, the second gene is a human gene. In another embodiment the second gene is a mouse or a rat gene.

In one embodiment, the second siRNA targets the XBP-1 gene. Exemplary siRNA targeting XBP-1 can be found in U.S. patent application Ser. No. 12/425,811 filed Apr. 17, 2009 (published as US 2009-0275638). The sequences of the target, sense, and antisense strands are incorporated by reference for all purposes.

Tables 3 and 9-13 disclose sequences of the target, sense strands, and antisense strands of XBP-1 targeting siRNA.

In one embodiment the first siRNA is AD-18038. The dsRNA AD-18038 targets the human XBP-1 gene at nucleotides 896-914 of a human XBP-1 gene (accession number NM_001004210).

TABLE 3 AD-18038 siRNA sequences SEQ ID Table 3: AD-18038 Sequence 5′ to 3′ NO: Target sequence CACCCUGAAUUCAUUGUCU 4153 Sense strand CACCCUGAAUUCAUUGUCU 4154 Sense strand, cAcccuGAAuucAuuGucudTsdT 4155 modified Antisense strand AGACAAUGAAUUCAGGGUG 4156 Antisense strand, AGAcAAUGAAUUcAGGGUGdTsdT 4157 modified

Additional dsRNA

A dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences in Tables 1-13, and differing in their ability to inhibit the expression of a target gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.

In addition, the RNAs provided in Tables 1-13 identify a site in the target gene transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a target gene.

While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, above represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in Tables 1-13, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.

An iRNA as described in Tables 1-13 can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described in Tables 1-13 contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of a PCSK9 gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a PCSK9 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a PCSK9 gene is important, especially if the particular region of complementarity in a PCSK9 gene is known to have polymorphic sequence variation within the population.

Covalent Linkage

The dual targeting siRNA agents of the invention include two siRNAs joined via a covalent linker. Covalent linkers are well-known to one of skill in the art and include, e.g., a nucleic acid linker, a peptide linker, and the like.

The covalent linker joins the two siRNAs. The covalent linker can join two sense strands, two antisense strands, one sense and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, or two sense and two antisense strands.

The covalent linker can include RNA and/or DNA and/or a peptide. The linker can be single stranded, double stranded, partially single strands, or partially double stranded. In some embodiments the linker includes a disulfide bond. The linker can be cleavable or non-cleavable.

The covalent linker can be, e.g., dTsdTuu=(5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate); rUsrU (a thiophosphate linker: 5′-uridyl-3′-thiophosphate-5′-uridyl-3′-phosphate); an rUrU linker; dTsdTaa (aadTsdT, 5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-adenyl-3′-phosphate-5′-adenyl-3′-phosphate); dTsdT (5′-2′deoxythyrnidyl-3′-thiophosphate-5′-2′ deoxythymidyl-3′-phosphate); dTsdTuu=uudTsdT=5′-2′deoxythymidyl-3′-thiophosphate-5′-2′deoxythymidyl-3′-phosphate-5′-uridyl-3′-phosphate-5′-uridyl-3′-phosphate.

The covalent linker can be a polyRNA, such as poly(5′-adenyl-3′-phosphate-AAAAAAAA) or poly(5′-cytidyl-3′-phosphate-5′-uridyl-3′-phosphate-CUCUCUCU)), e.g., X_(n) single stranded poly RNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyRNA linker. The covalent linker can be a polyDNA, such as poly(5′-2′deoxythymidyl-3′-phosphate-TTTTTTTT), e.g., wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker. a single stranded polyDNA linker wherein n is an integer from 2-50 inclusive, preferable 4-15 inclusive, most preferably 7-8 inclusive. Modified nucleotides or a mixture of nucleotides can also be present in said polyDNA linker.

The covalent linker can include a disulfide bond, optionally a bis-hexyl-disulfide linker. In one embodiment, the disulfide linker is

The covalent linker can include a peptide bond, e.g., include amino acids. In one embodiment, the covalent linker is a 1-10 amino acid long linker, preferably comprising 4-5 amino acids, optionally X-Gly-Phe-Gly-Y wherein X and Y represent any amino acid.

The covalent linker can include HEG, a hexaethylenglycol linker.

Modifications

In yet another embodiment, at least one of the siRNA of the dual targeting siRNA agent is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2′-docosanoyl-uridine-3′-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in U.S. Provisional Patent Application No. 61/223,665 (“the '665 application”), filed Jul. 7, 2009, entitled “Oligonucleotide End Caps” and International patent application no. PCT/US10/41214, filed Jul. 7, 2010.

Ligands Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In one ligand, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:4158). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:4159)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:4160)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:4161)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing α_(V)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference.

Chimeras

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Non-Ligand Groups

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

Vector Encoded dsRNAs

In another aspect, the dsRNAs of the invention can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (0 polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Pharmaceutical Compositions Containing iRNA

In one embodiment, the invention provides pharmaceutical compositions containing a dual targeting siRNA agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the siRNA is useful for treating a disease or disorder associated with the expression or activity of a target gene, such as pathological processes mediated by PCSK9 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of the target genes. In general, a suitable dose of siRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg, or 50 mg/kg per single dose.

The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose of siRNA on PCSK9 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by PCSK9 expression. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a transgene expressing human PCSK9.

The present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_(1215G), that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a dual targeting siRNA agent featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a nucleic acid-lipid particle, e.g., a SPLP, pSPLP, or SNALP. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. Nucleic acid-lipid particles, e.g., SNALPs, typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP”, which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683.

The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. For example, the mean diameter of the particles can be about 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 140 nm, 145 nm, or 150 nm.

In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The lipid to dsRNA ratio can be about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 113:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.

The nucleic acid lipid particles include a cationic lipid. The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (XTC), (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech Gl, e.g., C12-200), or a mixture thereof.

The cationic lipid may comprise from about 10 mol % to about 70 mol % or about 40 mol % of the total lipid present in the particle. The cationic lipid may comprise 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol % of the total lipid present in the particle. The cationic lipid may comprise 57.1 mol % or 57.5 mol % of the total lipid present in the particle.

In one embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (XTC) can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The nucleic acid lipid particle generally includes a non-cationic lipid. The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof.

The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. The non-cationic lipid may be about 5 mol %, 6 mol %, 7 mol %, 7.5 mol %, 7.7 mol %, 8 mol %, 9 mol %, 10 mol %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, 16 mol %, 17 mol %, 18 mol %, 19 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, 60 mol %, 65 mol %, 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90 mol %, or 95 mol %.

The nucleic acid lipid particle generally includes a conjugated lipid. The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid can be PEG-DMG (PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000); PEG-DSG (PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000); or PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000).

The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0 17.0, 18, 19.0 or 20.0 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. For example, the nucleic acid-lipid particle further includes cholesterol at about 5 mol %, 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol %. The nucleic acid-lipid particle can include cholesterol at about 31.5 mol %, 34.4 mol %, 35 mol %, 38.5 mol %, or 40 mol % of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Exemplary Nucleic Acid Lipid Particles

Additional exemplary lipid-dsRNA formulations are as follows:

TABLE A cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Cationic Mol % ratios Lipid Lipid:siRNA ratio SNALP DLinDMA DLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 S-XTC XTC XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 XTC XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 XTC XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 XTC XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 ALN100 ALN100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP11 MC3 MC-3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP12 C12-200 C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International patent application no. PCT/US10/22614, filed Jan. 29, 2010, which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are hereby incorporated by reference.

ALN100, i.e., ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200, i.e., Tech G1 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009, which is hereby incorporated by reference.

Synthesis of Cationic Lipids.

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry. —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

In one embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A; XTC is a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring.

In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃ and R₄ are independently lower alkyl or R₃ and R₄ can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]-232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an. Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR □=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6. Found 654.6.

General Synthesis of Nucleic Acid Lipid Particles

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Other Formulations

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target one or more of PD-1, PD-L1, or B7-H1 (CD80) (e.g., monoclonal antibodies against PD-1, PD-L1, or B7-H1), or one or more recombinant cytokines (e.g., IL6, IFN-γ, and TNF).

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the dual targeting siRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PCSK9 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Methods Using Dual Targeting siRNAs

In one aspect, the invention provides use of a dual targeting siRNA agent for inhibiting the expression of the PCSK9 gene in a mammal. The method includes administering a composition of the invention to the mammal such that expression of the target PCSK9 gene is decreased. In some embodiments, PCSK9 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer. For example, in certain instances, expression of the PCSK9 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a dual targeting siRNA agent described herein. In some embodiments, the PCSK9 gene is suppressed by at least about 60%, 70%, or 80% by administration of the dual targeting siRNA agent. In some embodiments, the PCSK9 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide.

The methods and compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating PCSK9 gene expression. For example, the compositions described herein can be used to treat hyperlipidemia and other forms of lipid imbalance such as hypercholesterolemia, hypertriglyceridemia and the pathological conditions associated with these disorders such as heart and circulatory diseases

Therefore, the invention also relates to the use of a dual targeting siRNA agent for the treatment of a PCSK9-mediated disorder or disease. For example, a dual targeting siRNA agent is used for treatment of a hyperlipidemia.

The effect of the decreased PCSK9 gene preferably results in a decrease in LDLc (low density lipoprotein cholesterol) levels in the blood, and more particularly in the serum, of the mammal. In some embodiments, LDLc levels are decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

The method includes administering a dual targeting siRNA agent to the subject to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some embodiments, the compositions are administered by intravenous infusion or injection.

The method includes administering a dual targeting siRNA agent, e.g., a dose sufficient to depress levels of PCSK9 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the PCSK9 gene in a subject.

In one embodiment, doses of dual targeting siRNA agent are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administrations can be maintained for one, two, three, or six months, or one year or longer.

In another embodiment, administration can be provided when Low Density Lipoprotein cholesterol (LDLc) levels reach or surpass a predetermined minimal level, such as greater than 70 mg/dL, 130 mg/dL, 150 mg/dL, 200 mg/dL, 300 mg/dL, or 400 mg/dL.

In general, the dual targeting siRNA agent does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-alpha or IFN-alpha levels. For example, when measured by an assay, such as an in vitro PBMC assay, such as described herein, the increase in levels of TNF-alpha or IFN-alpha, is less than 30%, 20%, or 10% of control cells treated with a control dsRNA such as a dsRNA that does not target PCSK9.

For example, a subject can be administered a therapeutic amount of dual targeting siRNA agent, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dual targeting siRNA agent can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the dual targeting siRNA agent can reduce PCSK9 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given dual targeting siRNA agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Additional Agents

In further embodiments, administration of a dual targeting siRNA agent is administered in combination an additional therapeutic agent. The dual targeting siRNA agent and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

Examples of additional therapeutic agents include those known to treat an agent known to treat a lipid disorders, such as hypercholesterolemia, atherosclerosis or dyslipidemia. For example, a dual targeting siRNA agent featured in the invention can be administered with, e.g., an HMG-CoA reductase inhibitor (e.g., a statin), a fibrate, a bile acid sequestrant, niacin, an antiplatelet agent, an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist (e.g., losartan potassium, such as Merck & Co.'s Cozaar®), an acylCoA cholesterol acetyltransferase (ACAT) inhibitor, a cholesterol absorption inhibitor, a cholesterol ester transfer protein (CETP) inhibitor, a microsomal triglyceride transfer protein (MTTP) inhibitor, a cholesterol modulator, a bile acid modulator, a peroxisome proliferation activated receptor (PPAR) agonist, a gene-based therapy, a composite vascular protectant (e.g., AGI-1067, from Atherogenics), a glycoprotein inhibitor, aspirin or an aspirin-like compound, an IBAT inhibitor (e.g., S-8921, from Shionogi), a squalene synthase inhibitor, or a monocyte chemoattractant protein (MCP)-I inhibitor. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Pfizer's Lipitor®/Tahor/Sortis/Torvast/Cardyl), pravastatin (Bristol-Myers Squibb's Pravachol, Sankyo's Mevalotin/Sanaprav), simvastatin (Merck's Zocor®/Sinvacor, Boehringer Ingelheim's Denan, Banyu's Lipovas), lovastatin (Merck's Mevacor/Mevinacor, Bexal's Lovastatina, Cepa; Schwarz Pharma's Liposcler), fluvastatin (Novartis' Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, e.g., bezafibrate (e.g., Roche's Befizal®/Cedur®/Bezalip®, Kissei's Bezatol), clofibrate (e.g., Wyeth's Atromid-S®), fenofibrate (e.g., Fournier's Lipidil/Lipantil, Abbott's Tricor®, Takeda's Lipantil, generics), gemfibrozil (e.g., Pfizer's Lopid/Lipur) and ciprofibrate (Sanofi-Synthelabo's Modalim®). Exemplary bile acid sequestrants include, e.g., cholestyramine (Bristol-Myers Squibb's Questran® and Questran Light™), colestipol (e.g., Pharmacia's Colestid), and colesevelam (Genzyme/Sankyo's WelChol™). Exemplary niacin therapies include, e.g., immediate release formulations, such as Aventis' Nicobid, Upsher-Smith's Niacor, Aventis' Nicolar, and Sanwakagaku's Perycit. Niacin extended release formulations include, e.g., Kos Pharmaceuticals' Niaspan and Upsher-Smith's SIo-Niacin. Exemplary antiplatelet agents include, e.g., aspirin (e.g., Bayer's aspirin), clopidogrel (Sanofi-Synthelabo/Bristol-Myers Squibb's Plavix), and ticlopidine (e.g., Sanofi-Synthelabo's Ticlid and Daiichi's Panaldine). Other aspirin-like compounds useful in combination with a dsRNA targeting PCSK9 include, e.g., Asacard (slow-release aspirin, by Pharmacia) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA). Exemplary angiotensin-converting enzyme inhibitors include, e.g., ramipril (e.g., Aventis' Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl CoA cholesterol acetyltransferase (ACAT) inhibitors include, e.g., avasimibe (Pfizer), eflucimibe (BioM{acute over (ε)}rieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, e.g., ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, e.g., Torcetrapib (also called CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, e.g., implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, e.g., NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst). Exemplary bile acid modulators include, e.g., HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferation activated receptor (PPAR) agonists include, e.g., tesaglitazar (AZ-242) (AstraZeneca), Netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene-based therapies include, e.g., AdGWEGF121.10 (GenVec), ApoAl (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-Al (ABCAl) (CV Therapeutics/Incyte, Aventis, Xenon). Exemplary Glycoprotein IIb/IIIa inhibitors include, e.g., roxifiban (also called DMP754, Bristol-Myers Squibb), Gantofiban (Merck KGaA/Yamanouchi), and Cromafiban (Millennium Pharmaceuticals). Exemplary squalene synthase inhibitors include, e.g., BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). An exemplary MCP-I inhibitor is, e.g., RS-504393 (Roche Bioscience). The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also appropriate for administering in combination with a dsRNA featured in the invention. Exemplary combination therapies suitable for administration with a dsRNA targeting PCSK9 include, e.g., advicor (Niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets by Merck/Schering-Plough Pharmaceuticals). Agents for treating hypercholesterolemia, and suitable for administration in combination with a dsRNA targeting PCSK9 include, e.g., lovastatin, niacin Altoprev® Extended-Release Tablets (Andrx Labs), lovastatin Caduet® Tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® Tablets (AstraZeneca), rosuvastatin calcium Lescol® Capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® Tablets (Parke-Davis), atorvastatin calcium Lofibra® Capsules (Gate), Niaspan Extended-Release Tablets (Kos), niacin Pravachol Tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® Tablets (Abbott), fenofibrate Vytorin® 10/10 Tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ Tablets (Sankyo), colesevelam hydrochloride Zetia® Tablets (Schering), ezetimibe Zetia® Tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor® Tablets (Merck).

In one embodiment, a dual targeting siRNA agent is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)).

In one embodiment, the dual targeting siRNA agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the dual targeting siRNA agent and the additional therapeutic agent are administered at the same time.

In another aspect, the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a dual targeting siRNA agent described herein. The method includes, optionally, providing the end user with one or more doses of the dual targeting siRNA agent, and instructing the end user to administer the dual targeting siRNA agent on a regimen described herein, thereby instructing the end user.

Identification of Patients

In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of LDL lowering, LDL lowering without lowering of HDL, ApoB lowering, or total cholesterol lowering. The method includes administering to the patient a dual targeting siRNA agent in an amount sufficient to lower the patient's LDL levels or ApoB levels, e.g., without substantially lowering HDL levels.

Genetic predisposition plays a role in the development of target gene associated diseases, e.g., hyperlipidemia. Therefore, a patient in need of a dual targeting siRNA agent can be identified by taking a family history, or, for example, screening for one or more genetic markers or variants. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a dual targeting siRNA agent. For example, a DNA test may also be performed on the patient to identify a mutation in the PCSK9 gene, before a PCSK9 dsRNA is administered to the patient.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Oligonucleotide Synthesis.

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to −30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.

Analysis

The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotides are diluted in water to 150 μL and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

iRNA Preparation

For the general preparation of iRNA, equimolar amounts of sense and antisense strand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.

Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table B.

TABLE B Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A adenosine C cytidine G guanosine U uridine N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u 2′-O-methyluridine dT, T 2′-deoxythymidine s phosphorothioate linkage

Example 2 PCSK9 siRNA Design, Synthesis, and Screening

A description of the design, synthesis, and assays using PCSK9 siRNA can be found in detail in U.S. patent application Ser. No. 11/746,864 filed on May 10, 2007 (now U.S. Pat. No. 7,605,251) and International Patent Application No. PCT/US2007/068655 filed May 10, 2007 (published as WO 2007/134161) and in U.S. patent application Ser. No. 12/478,452 filed Jun. 4, 2009 (published as US 2010/0010066) and International Patent Application No. PCT/US2009/032743 filed Jan. 30, 2009 (published as WO 2009/134487). All are incorporated by reference in their entirety for all purposes.

The sequences of siRNA targeting a PCSK9 gene are described in Table 1 and Table 2 above, and Tables 4-8 below.

Example 3 XBP-1 siRNA Design, Synthesis, and Screening

A description of the design, synthesis, and assays using XBP-1 siRNA can be found in detail in U.S. patent application Ser. No. 12/425,811 filed on Apr. 17, 2009 and published as US 2009-0275638. This application is incorporated by reference in its entirety for all purposes.

The sequences of siRNA targeting a XBP-1 gene are described in Table 3 above, and Tables 9-13 below.

Example 4 A Dual Targeting siRNA Agent

A dual targeting siRNA agent was synthesized. The sense and antisense strands for AD-10792 (target gene is PCSK9, see Table 2)) and AD-18038 (target gene is XBP-1, see Table 3) were synthesized. The two sense strands were covalently bound using a disulfide linker “Q51” with the structure shown below.

The resulting dual sense strand was hybridized to the corresponding antisense strands to create a 42 mer dual targeting siRNA agent “AD-23426” (SEQ ID NOS 4162-4165, respectively, in order of appearance):

GccuGGAGuuuAuucGGAAdTsdTQ51cAcccuGAAuucAuuGucudTsd TdTsdTCGGAcCUCAAAuAAGCCUU dTsdTGUGGGAcUUAAGUAAcAGA

Example 5 Inhibition of PCSK9 and Xbp-1 mRNA Levels by the PCSK9-Xbp1 Dual Targeting siRNA in Primary Mouse Hepatocytes

Primary mouse hepatocytes were transfected with dual targeting AD-23426 or individual siRNAs (AD-10792 and AD-18038) in lipofectamine 2000 (Invitrogen protocol). 48 hours after transfection cells were harvested and lysed. PCSK9, Xbp-1 and GAPDH transcripts were measured via bDNA in cell lysates prepared according to manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed.

As shown in FIG. 1, the dual targeting siRNA was at least as effective at inhibiting their corresponding target gene as the single siRNAs.

Example 6 Inhibition of PCSK9 and Xbp-1 mRNA Levels and Reduction of Total Serum Cholesterol by the PCSK9-Xbp1 Dual Targeting siRNA in Mice

The dual targeting AD-23426 was formulated in an LNP09 formulation: XTC/DSPC/Cholesterol/PEG-DMG in a % mol ratio of 50/10/38.5/1.5 with a lipid:siRNA ratio of about 10:1. The LNP09-AD-23426 was administered by tail vein injection into C57B6 mice at 6.0 mg/kg, 2.0 mg/kg and 0.6 mg/kg. LNP09 formulated single siRNAs (AD-10792 and AD-18038) were administered each at 3.0 mg/kg, 1.0 mg/kg and 0.3 mg/kg. Livers and plasma were harvested 72 hours post-injection (5 animals per group).

PCSK9, Xbp-1 and GAPDH transcript levels were measured via bDNA in livers prepared according to the manufacturer's protocol. PCSK9 to GAPDH or Xbp-1 to GAPDH ratios were normalized to control (luciferase) and graphed. The results are shown in FIG. 2.

Total cholesterol was measure in serum according to manufacturer's instructions using a cholesterol kit from WAKO TX.

The results demonstrate that the dual targeting siRNAs were at least as effective at inhibiting their corresponding target as single siRNAs in vivo. The results also show that the dual targeting construct has an additive effect compared to the single siRNAs at reducing total serum cholesterol.

Example 7 No Induction of IFN-α and TNF-α in HuPBMC

The effect of a dual targeting siRNA, AD-23426, on IFN-α and TNF-α in human PBMC was investigated.

Whole Blood anti-coagulated with Sodium Heparin was obtained from healthy donors at Research Blood Components, Inc (Boston, Mass.). Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll-Hypaque density centrifugation. Isolated PBMC were seeded at 1×10⁵ cells/well in 96 well plates and cultured in RPMI 1640 GlutaMax Medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen). siRNAs were transfected using DOTAP Transfection Reagent (Roche Applied Science). DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/transfection reagent complexes were incubated for 15 minutes at room temperature prior to being added to PBMC. siRNAs were transfected at final concentrations of 266 nM, 133 nM or 67 nM using 16 μg/ml, 8 μg/ml or 4 μg/ml DOTAP, respectively. The ratio of siRNA to DOTAP is 16.5 pmol/μg. Transfected PBMC were incubated at 37° C., 5% CO₂ for 24 hrs after which supernatants were harvested and stored at −80° C. until analysis. Quantitative cytokine analysis was done using commercially available Instant ELISA Kits for IFN-α (BMS216INST) and TNF-α (BMS223INST); both from Bender MedSystems (Vienna, Austria).

LNP09 and DOTAP formulated siRNAs were administered. Control siRNAs were AD-1730, AD-1955, AD-6248, AD-18889, AD-5048, and AD-18221. AD-10792: PCSK9 siRNA. AD-18038: XBP-1 siRNA.

The results are shown in FIG. 4. AD-23426 did not induce production of IFN-α and TNF-α, similar to the result obtained with the single target gene siRNAs. As expected, unmodified siRNAs (AD-5048 and AD-18889) induced production of both IFN-α and TNF-α. These results demonstrate that a dual targeting siRNA does not induce an immune response.

Example 8 Reduction of Total Serum Cholesterol with PCSK9-Xbp1 Dual Targeting siRNA Humans

A human subject is treated with a pharmaceutical composition, e.g., a nucleic acid-lipid particle having a dual targeting siRNA agent.

At time zero, a suitable first dose of the pharmaceutical composition is subcutaneously administered to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is evaluated, e.g., by measurement of total serum cholesterol. This measurement can be accompanied by a measurement of PCSK9 expression in said subject, and/or the products of the successful siRNA-targeting of PCSK9 mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

After treatment, the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.

Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended.

TABLE 4 Sequences of siRNA targeted to PCSK9 SEQ Antisense SEQ Sense strand ID strand ID *Target (5′-3′)¹ NO: (5′-3′)¹ NO: Duplex #    2-20 AGCGACGUCGAGGCGCUCATT 1 UGAGCGCCUCGAC 2 AD-15220 GUCGCUTT   15-33 CGCUCAUGGUUGCAGGCGGTT 3 CCGCCUGCAACCA 4 AD-15275 UGAGCGTT   16-34 GCUCAUGGUUGCAGGCGGGTT 5 CCCGCCUGCAACC 6 AD-15301 AUGAGCTT   30-48 GCGGGCGCCGCCGUUCAGUTT 7 ACUGAACGGCGGC 8 AD-15276 GCCCGCTT   31-49 CGGGCGCCGCCGUUCAGUUTT 9 AACUGAACGGCGG 10 AD-15302 CGCCCGTT   32-50 GGGCGCCGCCGUUCAGUUCTT 11 GAACUGAACGGCG 12 AD-15303 GCGCCCTT   40-58 CCGUUCAGUUCAGGGUCUGTT 13 CAGACCCUGAACU 14 AD-15221 GAACGGTT   43-61 UUCAGUUCAGGGUCUGAGCTT 15 GCUCAGACCCUGA 16 AD-15413 ACUGAATT   82-100 GUGAGACUGGCUCGGGCGGTT 17 CCGCCCGAGCCAG 18 AD-15304 UCUCACTT  100-118 GGCCGGGACGCGUCGUUGCTT 19 GCAACGACGCGUC 20 AD-15305 CCGGCCTT  101-119 GCCGGGACGCGUCGUUGCATT 21 UGCAACGACGCGU 22 AD-15306 CCCGGCTT  102-120 CCGGGACGCGUCGUUGCAGTT 23 CUGCAACGACGCG 24 AD-15307 UCCCGGTT  105-123 GGACGCGUCGUUGCAGCAGTT 25 CUGCUGCAACGAC 26 AD-15277 GCGUCCTT  135-153 UCCCAGCCAGGAUUCCGCGTsT 27 CGCGGAAUCCUGG 28 AD-9526 CUGGGATsT  135-153 ucccAGccAGGAuuccGcGTsT 29 CGCGGAAUCCUGG 30 AD-9652 CUGGGATsT  136-154 CCCAGCCAGGAUUCCGCGCTsT 31 GCGCGGAAUCCUG 32 AD-9519 GCUGGGTsT  136-154 cccAGccAGGAuuccGcGcTsT 33 GCGCGGAAUCCUG 34 AD-9645 GCUGGGTsT  138-156 CAGCCAGGAUUCCGCGCGCTsT 35 GCGCGCGGAAUCC 36 AD-9523 UGGCUGTsT  138-156 cAGccAGGAuuccGcGcGcTsT 37 GCGCGCGGAAUCC 38 AD-9649 UGGCUGTsT  185-203 AGCUCCUGCACAGUCCUCCTsT 39 GGAGGACUGUGCA 40 AD-9569 GGAGCUTsT  185-203 AGcuccuGcAcAGuccuccTsT 41 GGAGGACUGUGcA 42 AD-9695 GGAGCUTsT  205-223 CACCGCAAGGCUCAAGGCGTT 43 CGCCUUGAGCCUU 44 AD-15222 GCGGUGTT  208-226 CGCAAGGCUCAAGGCGCCGTT 45 CGGCGCCUUGAGC 46 AD-15278 CUUGCGTT  210-228 CAAGGCUCAAGGCGCCGCCTT 47 GGCGGCGCCUUGA 48 AD-15178 GCCUUGTT  232-250 GUGGACCGCGCACGGCCUCTT 49 GAGGCCGUGCGCG 50 AD-15308 GUCCACTT  233-251 UGGACCGCGCACGGCCUCUTT 51 AGAGGCCGUGCGC 52 AD-15223 GGUCCATT  234-252 GGACCGCGCACGGCCUCUATT 53 UAGAGGCCGUGCG 54 AD-15309 CGGUCCTT  235-253 GACCGCGCACGGCCUCUAGTT 55 CUAGAGGCCGUGC 56 AD-15279 GCGGUCTT  236-254 ACCGCGCACGGCCUCUAGGTT 57 CCUAGAGGCCGUG 58 AD-15194 CGCGGUTT  237-255 CCGCGCACGGCCUCUAGGUTT 59 ACCUAGAGGCCGU 60 AD-15310 GCGCGGTT  238-256 CGCGCACGGCCUCUAGGUCTT 61 GACCUAGAGGCCG 62 AD-15311 UGCGCGTT  239-257 GCGCACGGCCUCUAGGUCUTT 63 AGACCUAGAGGCC 64 AD-15392 GUGCGCTT  240-258 CGCACGGCCUCUAGGUCUCTT 65 GAGACCUAGAGGC 66 AD-15312 CGUGCGTT  248-266 CUCUAGGUCUCCUCGCCAGTT 67 CUGGCGAGGAGAC 68 AD-15313 CUAGAGTT  249-267 UCUAGGUCUCCUCGCCAGGTT 69 CCUGGCGAGGAGA 70 AD-15280 CCUAGATT  250-268 CUAGGUCUCCUCGCCAGGATT 71 UCCUGGCGAGGAG 72 AD-15267 ACCUAGTT  252-270 AGGUCUCCUCGCCAGGACATT 73 UGUCCUGGCGAGG 74 AD-15314 AGACCUTT  258-276 CCUCGCCAGGACAGCAACCTT 75 GGUUGCUGUCCUG 76 AD-15315 GCGAGGTT  300-318 CGUCAGCUCCAGGCGGUCCTsT 77 GGACCGCCUGGAG 78 AD-9624 CUGACGTsT  300-318 cGucAGcuccAGGcGGuccTsT 79 GGACCGCCUGGAG 80 AD-9750 CUGACGTsT  301-319 GUCAGCUCCAGGCGGUCCUTsT 81 AGGACCGCCUGGA 82 AD-9623 GCUGACTsT  301-319 GucAGcuccAGGcGGuccuTsT 83 AGGACCGCCUGGA 84 AD-9749 GCUGACTsT  370-388 GGCGCCCGUGCGCAGGAGGTT 85 CCUCCUGCGCACG 86 AD-15384 GGCGCCTT  408-426 GGAGCUGGUGCUAGCCUUGTsT 87 CAAGGCUAGCACC 88 AD-9607 AGCUCCTsT  408-426 GGAGcuGGuGcuAGccuuGTsT 89 cAAGGCuAGcACc 90 AD-9733 AGCUCCTsT  411-429 GCUGGUGCUAGCCUUGCGUTsT 91 ACGCAAGGCUAGC 92 AD-9524 ACCAGCTsT  411-429 GcuGGuGcuAGccuuGcGuTsT 93 ACGcAAGGCuAGc 94 AD-9650 ACcAGCTsT  412-430 CUGGUGCUAGCCUUGCGUUTsT 95 AACGCAAGGCUAG 96 AD-9520 CACCAGTsT  412-430 CUGGUGCUAGCCUUGCGUUTsT 97 AACGCAAGGCUAG 98 AD-9520 CACCAGTsT  412-430 cuGGuGcuAGccuuGcGuuTsT 99 AACGcAAGGCuAG 100 AD-9646 cACcAGTsT  416-434 UGCUAGCCUUGCGUUCCGATsT 101 UCGGAACGCAAGG 102 AD-9608 CUAGCATsT  416-434 uGcuAGccuuGcGuuccGATsT 103 UCGGAACGcAAGG 104 AD-9734 CuAGcATsT  419-437 UAGCCUUGCGUUCCGAGGATsT 105 UCCUCGGAACGCA 106 AD-9546 AGGCUATsT  419-437 uAGccuuGcGuuccGAGGATsT 107 UCCUCGGAACGcA 108 AD-9672 AGGCuATsT  439-457 GACGGCCUGGCCGAAGCACTT 109 GUGCUUCGGCCAG 110 AD-15385 GCCGUCTT  447-465 GGCCGAAGCACCCGAGCACTT 111 GUGCUCGGGUGCU 112 AD-15393 UCGGCCTT  448-466 GCCGAAGCACCCGAGCACGTT 113 CGUGCUCGGGUGC 114 AD-15316 UUCGGCTT  449-467 CCGAAGCACCCGAGCACGGTT 115 CCGUGCUCGGGUG 116 AD-15317 CUUCGGTT  458-476 CCGAGCACGGAACCACAGCTT 117 GCUGUGGUUCCGU 118 AD-15318 GCUCGGTT  484-502 CACCGCUGCGCCAAGGAUCTT 119 GAUCCUUGGCGCA 120 AD-15195 GCGGUGTT  486-504 CCGCUGCGCCAAGGAUCCGTT 121 CGGAUCCUUGGCG 122 AD-15224 CAGCGGTT  487-505 CGCUGCGCCAAGGAUCCGUTT 123 ACGGAUCCUUGGC 124 AD-15188 GCAGCGTT  489-507 CUGCGCCAAGGAUCCGUGGTT 125 CCACGGAUCCUUG 126 AD-15225 GCGCAGTT  500-518 AUCCGUGGAGGUUGCCUGGTT 127 CCAGGCAACCUCC 128 AD-15281 ACGGAUTT  509-527 GGUUGCCUGGCACCUACGUTT 129 ACGUAGGUGCCAG 130 AD-15282 GCAACCTT  542-560 AGGAGACCCACCUCUCGCATT 131 UGCGAGAGGUGGG 132 AD-15319 UCUCCUTT  543-561 GGAGACCCACCUCUCGCAGTT 133 CUGCGAGAGGUGG 134 AD-15226 GUCUCCTT  544-562 GAGACCCACCUCUCGCAGUTT 135 ACUGCGAGAGGUG 136 AD-15271 GGUCUCTT  549-567 CCACCUCUCGCAGUCAGAGTT 137 CUCUGACUGCGAG 138 AD-15283 AGGUGGTT  552-570 CCUCUCGCAGUCAGAGCGCTT 139 GCGCUCUGACUGC 140 AD-15284 GAGAGGTT  553-571 CUCUCGCAGUCAGAGCGCATT 141 UGCGCUCUGACUG 142 AD-15189 CGAGAGTT  554-572 UCUCGCAGUCAGAGCGCACTT 143 GUGCGCUCUGACU 144 AD-15227 GCGAGATT  555-573 CUCGCAGUCAGAGCGCACUTsT 145 AGUGCGCUCUGAC 146 AD-9547 UGCGAGTsT  555-573 cucGcAGucAGAGcGcAcuTsT 147 AGUGCGCUCUGAC 148 AD-9673 UGCGAGTsT  558-576 GCAGUCAGAGCGCACUGCCTsT 149 GGCAGUGCGCUCU 150 AD-9548 GACUGCTsT  558-576 GcAGucAGAGcGcAcuGccTsT 151 GGcAGUGCGCUCU 152 AD-9674 GACUGCTsT  606-624 GGGAUACCUCACCAAGAUCTsT 153 GAUCUUGGUGAGG 154 AD-9529 UAUCCCTsT  606-624 GGGAuAccucAccAAGAucTsT 155 GAUCUUGGUGAGG 156 AD-9655 uAUCCCTsT  659-677 UGGUGAAGAUGAGUGGCGATsT 157 UCGCCACUCAUCU 158 AD-9605 UCACCATsT  659-677 uGGuGAAGAuGAGuGGcGATsT 159 UCGCcACUcAUCU 160 AD-9731 UcACcATsT  663-681 GAAGAUGAGUGGCGACCUGTsT 161 CAGGUCGCCACUC 162 AD-9596 AUCUUCTsT  663-681 GAAGAuGAGuGGcGAccuGTsT 163 cAGGUCGCcACUc 164 AD-9722 AUCUUCTsT  704-722 CCCAUGUCGACUACAUCGATsT 165 UCGAUGUAGUCGA 166 AD-9583 CAUGGGTsT  704-722 cccAuGucGAcuAcAucGATsT 167 UCGAUGuAGUCGA 168 AD-9709 cAUGGGTsT  718-736 AUCGAGGAGGACUCCUCUGTsT 169 CAGAGGAGUCCUC 170 AD-9579 CUCGAUTsT  718-736 AucGAGGAGGAcuccucuGTsT 171 cAGAGGAGUCCUC 172 AD-9705 CUCGAUTsT  758-776 GGAACCUGGAGCGGAUUACTT 173 GUAAUCCGCUCCA 174 AD-15394 GGUUCCTT  759-777 GAACCUGGAGCGGAUUACCTT 175 GGUAAUCCGCUCC 176 AD-15196 AGGUUCTT  760-778 AACCUGGAGCGGAUUACCCTT 177 GGGUAAUCCGCUC 178 AD-15197 CAGGUUTT  777-795 CCCUCCACGGUACCGGGCGTT 179 CGCCCGGUACCGU 180 AD-15198 GGAGGGTT  782-800 CACGGUACCGGGCGGAUGATsT 181 UCAUCCGCCCGGU 182 AD-9609 ACCGUGTsT  782-800 cAcGGuAccGGGcGGAuGATsT 183 UcAUCCGCCCGGu 184 AD-9735 ACCGUGTsT  783-801 ACGGUACCGGGCGGAUGAATsT 185 UUCAUCCGCCCGG 186 AD-9537 UACCGUTsT  783-801 AcGGuAccGGGcGGAuGAATsT 187 UUcAUCCGCCCGG 188 AD-9663 uACCGUTsT  784-802 CGGUACCGGGCGGAUGAAUTsT 189 AUUCAUCCGCCCG 190 AD-9528 GUACCGTsT  784-802 cGGuAccGGGcGGAuGAAuTsT 191 AUUcAUCCGCCCG 192 AD-9654 GuACCGTsT  785-803 GGUACCGGGCGGAUGAAUATsT 193 UAUUCAUCCGCCC 194 AD-9515 GGUACCTsT  785-803 GGuAccGGGcGGAuGAAuATsT 195 uAUUcAUCCGCCC 196 AD-9641 GGuACCTsT  786-804 GUACCGGGCGGAUGAAUACTsT 197 GUAUUCAUCCGCC 198 AD-9514 CGGUACTsT  786-804 GuAccGGGcGGAuGAAuAcTsT 199 GuAUUcAUCCGCC 200 AD-9640 CGGuACTsT  788-806 ACCGGGCGGAUGAAUACCATsT 201 UGGUAUUCAUCCG 202 AD-9530 CCCGGUTsT  788-806 AccGGGcGGAuGAAuAccATsT 203 UGGuAUUcAUCCG 204 AD-9656 CCCGGUTsT  789-807 CCGGGCGGAUGAAUACCAGTsT 205 CUGGUAUUCAUCC 206 AD-9538 GCCCGGTsT  789-807 ccGGGcGGAuGAAuAccAGTsT 207 CUGGuAUUcAUCC 208 AD-9664 GCCCGGTsT  825-843 CCUGGUGGAGGUGUAUCUCTsT 209 GAGAUACACCUCC 210 AD-9598 ACCAGGTsT  825-843 ccuGGuGGAGGuGuAucucTsT 211 GAGAuAcACCUCc 212 AD-9724 ACcAGGTsT  826-844 CUGGUGGAGGUGUAUCUCCTsT 213 GGAGAUACACCUC 214 AD-9625 CACCAGTsT  826-844 cuGGuGGAGGuGuAucuccTsT 215 GGAGAuAcACCUC 216 AD-9751 cACcAGTsT  827-845 UGGUGGAGGUGUAUCUCCUTsT 217 AGGAGAUACACCU 218 AD-9556 CCACCATsT  827-845 uGGuGGAGGuGuAucuccuTsT 219 AGGAGAuAcACCU 220 AD-9682 CcACcATsT  828-846 GGUGGAGGUGUAUCUCCUATsT 221 UAGGAGAUACACC 222 AD-9539 UCCACCTsT  828-846 GGuGGAGGuGuAucuccuATsT 223 uAGGAGAuAcACC 224 AD-9665 UCcACCTsT  831-849 GGAGGUGUAUCUCCUAGACTsT 225 GUCUAGGAGAUAC 226 AD-9517 ACCUCCTsT  831-849 GGAGGuGuAucuccuAGAcTsT 227 GUCuAGGAGAuAc 228 AD-9643 ACCUCCTsT  833-851 AGGUGUAUCUCCUAGACACTsT 229 GUGUCUAGGAGAU 230 AD-9610 ACACCUTsT  833-851 AGGuGuAucuccuAGAcAcTsT 231 GUGUCuAGGAGAu 232 AD-9736 AcACCUTsT  833-851 AfgGfuGfuAfuCfuCfcUfaG 233 P*gUfgUfcUfaG 234 AD-14681 faCfaCfTsT fgAfgAfuAfcAf cCfuTsT  833-851 AGGUfGUfAUfCfUfCfCfUfA 235 GUfGUfCfUfAGG 236 AD-14691 GACfACfTsT AGAUfACfACfCf UfTsT  833-851 AgGuGuAuCuCcUaGaCaCTsT 237 P*gUfgUfcUfaG 238 AD-14701 fgAfgAfuAfcAf cCfuTsT  833-851 AgGuGuAuCuCcUaGaCaCTsT 239 GUfGUfCfUfAGG 240 AD-14711 AGAUfACfACfCf UfTsT  833-851 AfgGfuGfuAfuCfuCfcUfaG 241 GUGUCuaGGagAU 242 AD-14721 faCfaCfTsT ACAccuTsT  833-851 AGGUfGUfAUfCfUfCfCfUfA 243 GUGUCuaGGagAU 244 AD-14731 GACfACfTsT ACAccuTsT  833-851 AgGuGuAuCuCcUaGaCaCTsT 245 GUGUCuaGGagAU 246 AD-14741 ACAccuTsT  833-851 GfcAfcCfcUfcAfuAfgGfcC 247 P*uCfcAfgGfcC 248 AD-15087 fuGfgAfTsT fuAfuGfaGfgGf uGfcTsT  833-851 GCfACfCfCfUfCfAUfAGGCf 249 UfCfCfAGGCfCf 250 AD-15097 CfUfGGATsT UfAUfGAGGGUfG CfTsT  833-851 GcAcCcUcAuAgGcCuGgATsT 251 P*uCfcAfgGfcC 252 AD-15107 fuAfuGfaGfgGf uGfcTsT  833-851 GcAcCcUcAuAgGcCuGgATsT 253 UfCfCfAGGCfCf 254 AD-15117 UfAUfGAGGGUfG CfTsT  833-851 GfcAfcCfcUfcAfuAfgGfcC 255 UCCAGgcCUauGA 256 AD-15127 fuGfgAfTsT GGGugcTsT  833-851 GCfACfCfCfUfCfAUfAGGCf 257 UCCAGgcCUauGA 258 AD-15137 CfUfGGATsT GGGugcTsT  833-851 GcAcCcUcAuAgGcCuGgATsT 259 UCCAGgcCUauGA 260 AD-15147 GGGugcTsT  836-854 UGUAUCUCCUAGACACCAGTsT 261 CUGGUGUCUAGGA 262 AD-9516 GAUACATsT  836-854 uGuAucuccuAGAcAccAGTsT 263 CUGGUGUCuAGGA 264 AD-9642 GAuAcATsT  840-858 UCUCCUAGACACCAGCAUATsT 265 UAUGCUGGUGUCU 266 AD-9562 AGGAGATsT  840-858 ucuccuAGAcAccAGcAuATsT 267 uAUGCUGGUGUCu 268 AD-9688 AGGAGATsT  840-858 UfcUfcCfuAfgAfcAfcCfaG 269 P*uAfuGfcUfgG 270 AD-14677 fcAfuAfTsT fuGfuCfuAfgGf aGfaTsT  840-858 UfCfUfCfCfUfAGACfACfCf 271 UfAUfGCfUfGGU 272 AD-14687 AGCfAUfATsT fGUfCfUfAGGAG ATsT  840-858 UcUcCuAgAcAcCaGcAuATsT 273 P*uAfuGfcUfgG 274 AD-14697 fuGfuCfuAfgGf aGfaTsT  840-858 UcUcCuAgAcAcCaGcAuATsT 275 UfAUfGCfUfGGU 276 AD-14707 fGUfCfUfAGGAG ATsT  840-858 UfcUfcCfuAafAfcAfcCfaG 277 UAUGCugGUguCU 278 AD-14717 fcAfuAfTsT AGGagaTsT  840-858 UfCfUfCfCfUfAGACfACfCf 279 UAUGCugGUguCU 280 AD-14727 AGCfAUfATsT AGGagaTsT  840-858 UcUcCuAgAcAcCaGcAuATsT 281 UAUGCugGUguCU 282 AD-14737 AGGagaTsT  840-858 AfgGfcCfuGfgAfgUfuUfaU 283 P*cCfgAfaUfaA 284 AD-15083 fuCfgGfTsT faCfuCfcAfgGf cCfuTsT  840-858 AGGCfCfUfGGAGUfUfUfAUf 285 CfCfGAAUfAAAC 286 AD-15093 UfCfGGTsT fUfCfCfAGGCfC fUfTsT  840-858 AgGcCuGgAgUuUaUuCgGTsT 287 P*cCfgAfaUfaA 288 AD-15103 faCfuCfcAfgGf cCfuTsT  840-858 AgGcCuGgAgUuUaUuCgGTsT 289 CfCfGAAUfAAAC 290 AD-15113 fUfCfCfAGGCfC fUfTsT  840-858 AfgGfcCfuGfgAfgUfuUfaU 291 CCGAAuaAAcuCC 292 AD-15123 fuCfgGfTsT AGGccuTsT  840-858 AGGCfCfUfGGAGUfUfUfAUf 293 CCGAAuaAAcuCC 294 AD-15133 UfCfGGTsT AGGccuTsT  840-858 AgGcCuGgAgUuUaUuCgGTsT 295 CCGAAuaAAcuCC 296 AD-15143 AGGccuTsT  841-859 CUCCUAGACACCAGCAUACTsT 297 GUAUGCUGGUGUC 298 AD-9521 UAGGAGTsT  841-859 cuccuAGAcAccAGcAuAcTsT 299 GuAUGCUGGUGUC 300 AD-9647 uAGGAGTsT  842-860 UCCUAGACACCAGCAUACATsT 301 UGUAUGCUGGUGU 302 AD-9611 CUAGGATsT  842-860 uccuAGAcAccAGcAuAcATsT 303 UGuAUGCUGGUGU 304 AD-9737 CuAGGATsT  843-861 CCUAGACACCAGCAUACAGTsT 305 CUGUAUGCUGGUG 306 AD-9592 UCUAGGTsT  843-861 ccuAGAcAccAGcAuAcAGTsT 307 CUGuAUGCUGGUG 308 AD-9718 UCuAGGTsT  847-865 GACACCAGCAUACAGAGUGTsT 309 CACUCUGUAUGCU 310 AD-9561 GGUGUCTsT  847-865 GAcAccAGcAuAcAGAGuGTsT 311 cACUCUGuAUGCU 312 AD-9687 GGUGUCTsT  855-873 CAUACAGAGUGACCACCGGTsT 313 CCGGUGGUCACUC 314 AD-9636 UGUAUGTsT  855-873 cAuAcAGAGuGAccAccGGTsT 315 CCGGUGGUcACUC 316 AD-9762 UGuAUGTsT  860-878 AGAGUGACCACCGGGAAAUTsT 317 AUUUCCCGGUGGU 318 AD-9540 CACUCUTsT  860-878 AGAGuGAccAccGGGAAAuTsT 319 AUUUCCCGGUGGU 320 AD-9666 cACUCUTsT  861-879 GAGUGACCACCGGGAAAUCTsT 321 GAUUUCCCGGUGG 322 AD-9535 UCACUCTsT  861-879 GAGuGAccAccGGGAAAucTsT 323 GAUUUCCCGGUGG 324 AD-9661 UcACUCTsT  863-881 GUGACCACCGGGAAAUCGATsT 325 UCGAUUUCCCGGU 326 AD-9559 GGUCACTsT  863-881 GuGAccAccGGGAAAucGATsT 327 UCGAUUUCCCGGU 328 AD-9685 GGUcACTsT  865-883 GACCACCGGGAAAUCGAGGTsT 329 CCUCGAUUUCCCG 330 AD-9533 GUGGUCTsT  865-883 GAccAccGGGAAAucGAGGTsT 331 CCUCGAUUUCCCG 332 AD-9659 GUGGUCTsT  866-884 ACCACCGGGAAAUCGAGGGTsT 333 CCCUCGAUUUCCC 334 AD-9612 GGUGGUTsT  866-884 AccAccGGGAAAucGAGGGTsT 335 CCCUCGAUUUCCC 336 AD-9738 GGUGGUTsT  867-885 CCACCGGGAAAUCGAGGGCTsT 337 GCCCUCGAUUUCC 338 AD-9557 CGGUGGTsT  867-885 ccAccGGGAAAucGAGGGcTsT 339 GCCCUCGAUUUCC 340 AD-9683 CGGUGGTsT  875-893 AAAUCGAGGGCAGGGUCAUTsT 341 AUGACCCUGCCCU 342 AD-9531 CGAUUUTsT  875-893 AAAucGAGGGcAGGGucAuTsT 343 AUGACCCUGCCCU 344 AD-9657 CGAUUUTsT  875-893 AfaAfuCfgAfgGfgCfaGfgG 345 P*aUfgAfcCfcU 346 AD-14673 fuCfaUfTsT fgCfcCfuCfgAf uUfuTsT  875-893 AAAUfCfGAGGGCfAGGGUfCf 347 AUfGACfCfCfUf 348 AD-14683 AUfTsT GCfCfCfUfCfGA UfUfUfTsT  875-893 AaAuCgAgGgCaGgGuCaUTsT 349 P*aUfgAfcCfcU 350 AD-14693 fgCfcCfuCfgAf uUfuTsT  875-893 AaAuCgAgGgCaGgGuCaUTsT 351 AUfGACfCfCfUf 352 AD-14703 GCfCfCfUfCfGA UfUfUfTsT  875-893 AfaAfuCfgAfgGfgCfaGfgG 353 AUGACccUGccCU 354 AD-14713 fuCfaUfTsT CGAuuuTsT  875-893 AAAUfCfGAGGGCfAGGGUfCf 355 AUGACccUGccCU 356 AD-14723 AUfTsT CGAuuuTsT  875-893 AaAuCgAgGgCaGgGuCaUTsT 357 AUGACccUGccCU 358 AD-14733 CGAuuuTsT  875-893 CfgGfcAfcCfcUfcAfuAfgG 359 P*cAfgGfcCfuA 360 AD-15079 fcCfuGfTsT fuGfaGfgGfuGf cCfgTsT  875-893 CfGGCfACfCfCfUfCfAUfAG 361 CfAGGCfCfUfAU 362 AD-15089 GCfCfUfGTsT fGAGGGUfGCfCf GTsT  875-893 CgGcAcCcUcAuAgGcCuGTsT 363 P*cAfgGfcCfuA 364 AD-15099 fuGfaGfgGfuGf cCfgTsT  875-893 CgGcAcCcUcAuAgGcCuGTsT 365 CfAGGCfCfUfAU 366 AD-15109 fGAGGGUfGCfCf GTsT  875-893 CfgGfcAfcCfcUfcAfuAfgG 367 CAGGCcuAUgaGG 368 AD-15119 fcCfuGfTsT GUGccgTsT  875-893 CfGGCfACfCfCfUfCfAUfAG 369 CAGGCcuAUgaGG 370 AD-15129 GCfCfUfGTsT GUGccgTsT  875-893 CgGcAcCcUcAuAgGcCuGTsT 371 CAGGCcuAUgaGG 372 AD-15139 GUGccgTsT  877-895 AUCGAGGGCAGGGUCAUGGTsT 373 CCAUGACCCUGCC 374 AD-9542 CUCGAUTsT  877-895 AucGAGGGcAGGGucAuGGTsT 375 CcAUGACCCUGCC 376 AD-9668 CUCGAUTsT  878-896 cGAGGGcAGGGucAuGGucTsT 377 GACcAUGACCCUG 378 AD-9739 CCCUCGTsT  880-898 GAGGGCAGGGUCAUGGUCATsT 379 UGACCAUGACCCU 380 AD-9637 GCCCUCTsT  880-898 GAGGGcAGGGucAuGGucATsT 381 UGACcAUGACCCU 382 AD-9763 GCCCUCTsT  882-900 GGGCAGGGUCAUGGUCACCTsT 383 GGUGACCAUGACC 384 AD-9630 CUGCCCTsT  882-900 GGGcAGGGucAuGGucAccTsT 385 GGUGACcAUGACC 386 AD-9756 CUGCCCTsT  885-903 CAGGGUCAUGGUCACCGACTsT 387 GUCGGUGACCAUG 388 AD-9593 ACCCUGTsT  885-903 cAGGGucAuGGucAccGAcTsT 389 GUCGGUGACcAUG 390 AD-9719 ACCCUGTsT  886-904 AGGGUCAUGGUCACCGACUTsT 391 AGUCGGUGACCAU 392 AD-9601 GACCCUTsT  886-904 AGGGucAuGGucAccGAcuTsT 393 AGUCGGUGACcAU 394 AD-9727 GACCCUTsT  892-910 AUGGUCACCGACUUCGAGATsT 395 UCUCGAAGUCGGU 396 AD-9573 GACCAUTsT  892-910 AuGGucAccGAcuucGAGATsT 397 UCUCGAAGUCGGU 398 AD-9699 GACcAUTsT  899-917 CCGACUUCGAGAAUGUGCCTT 399 GGCACAUUCUCGA 400 AD-15228 AGUCGGTT  921-939 GGAGGACGGGACCCGCUUCTT 401 GAAGCGGGUCCCG 402 AD-15395 UCCUCCTT  993- CAGCGGCCGGGAUGCCGGCTsT 403 GCCGGCAUCCCGG 404 AD-9602 1011 CCGCUGTsT  993- cAGcGGccGGGAuGccGGcTsT 405 GCCGGcAUCCCGG 406 AD-9728 1011 CCGCUGTsT 1020- GGGUGCCAGCAUGCGCAGCTT 407 GCUGCGCAUGCUG 408 AD-15386 1038 GCACCCTT 1038- CCUGCGCGUGCUCAACUGCTsT 409 GCAGUUGAGCACG 410 AD-9580 1056 CGCAGGTsT 1038- ccuGcGcGuGcucAAcuGcTsT 411 GcAGUUGAGcACG 412 AD-9706 1056 CGcAGGTsT 1040- UGCGCGUGCUCAACUGCCATsT 413 UGGCAGUUGAGCA 414 AD-9581 1058 CGCGCATsT 1040- uGcGcGuGcucAAcuGccATsT 415 UGGcAGUUGAGcA 416 AD-9707 1058 CGCGcATsT 1042- CGCGUGCUCAACUGCCAAGTsT 417 CUUGGCAGUUGAG 418 AD-9543 1060 CACGCGTsT 1042- cGcGuGcucAAcuGccAAGTsT 419 CUUGGcAGUUGAG 420 AD-9669 1060 cACGCGTsT 1053- CUGCCAAGGGAAGGGCACGTsT 421 CGUGCCCUUCCCU 422 AD-9574 1071 UGGCAGTsT 1053- cuGccAAGGGAAGGGcAcGTsT 423 CGUGCCCUUCCCU 424 AD-9700 1071 UGGcAGTsT 1057- CAAGGGAAGGGCACGGUUATT 425 UAACCGUGCCCUU 426 AD-15320 1075 CCCUUGTT 1058- AAGGGAAGGGCACGGUUAGTT 427 CUAACCGUGCCCU 428 AD-15321 1076 UCCCUUTT 1059- AGGGAAGGGCACGGUUAGCTT 429 GCUAACCGUGCCC 430 AD-15199 1077 UUCCCUTT 1060- GGGAAGGGCACGGUUAGCGTT 431 CGCUAACCGUGCC 432 AD-15167 1078 CUUCCCTT 1061- GGAAGGGCACGGUUAGCGGTT 433 CCGCUAACCGUGC 434 AD-15164 1079 CCUUCCTT 1062- GAAGGGCACGGUUAGCGGCTT 435 GCCGCUAACCGUG 436 AD-15166 1080 CCCUUCTT 1063- AAGGGCACGGUUAGCGGCATT 437 UGCCGCUAACCGU 438 AD-15322 1081 GCCCUUTT 1064- AGGGCACGGUUAGCGGCACTT 439 GUGCCGCUAACCG 440 AD-15200 1082 UGCCCUTT 1068- CACGGUUAGCGGCACCCUCTT 441 GAGGGUGCCGCUA 442 AD-15213 1086 ACCGUGTT 1069- ACGGUUAGCGGCACCCUCATT 443 UGAGGGUGCCGCU 444 AD-15229 1087 AACCGUTT 1072- GUUAGCGGCACCCUCAUAGTT 445 CUAUGAGGGUGCC 446 AD-15215 1090 GCUAACTT 1073- UUAGCGGCACCCUCAUAGGTT 447 CCUAUGAGGGUGC 448 AD-15214 1091 CGCUAATT 1076- GCGGCACCCUCAUAGGCCUTsT 449 AGGCCUAUGAGGG 450 AD-9315 1094 UGCCGCTsT 1079- GCACCCUCAUAGGCCUGGATsT 451 UCCAGGCCUAUGA 452 AD-9326 1097 GGGUGCTsT 1085- UCAUAGGCCUGGAGUUUAUTsT 453 AUAAACUCCAGGC 454 AD-9318 1103 CUAUGATsT 1090- GGCCUGGAGUUUAUUCGGATsT 455 UCCGAAUAAACUC 456 AD-9323 1108 CAGGCCTsT 1091- GCCUGGAGUUUAUUCGGAATsT 457 UUCCGAAUAAACU 458 AD-9314 1109 CCAGGCTsT 1091- GccuGGAGuuuAuucGGAATsT 459 UUCCGAAuAAACU 460 AD-10792 1109 CcAGGCTsT 1091- GccuGGAGuuuAuucGGAATsT 461 UUCCGAAUAACUC 462 AD-10796 1109 CAGGCTsT 1093- CUGGAGUUUAUUCGGAAAATsT 463 UUUUCCGAAUAAA 464 AD-9638 1111 CUCCAGTsT 1093- cuGGAGuuuAuucGGAAAATsT 465 UUUUCCGAAuAAA 466 AD-9764 1111 CUCcAGTsT 1095- GGAGUUUAUUCGGAAAAGCTsT 467 GCUUUUCCGAAUA 468 AD-9525 1113 AACUCCTsT 1095- GGAGuuuAuucGGAAAAGcTsT 469 GCUUUUCCGAAuA 470 AD-9651 1113 AACUCCTsT 1096- GAGUUUAUUCGGAAAAGCCTsT 471 GGCUUUUCCGAAU 472 AD-9560 1114 AAACUCTsT 1096- GAGuuuAuucGGAAAAGccTsT 473 GGCUUUUCCGAAu 474 AD-9686 1114 AAACUCTsT 1100- UUAUUCGGAAAAGCCAGCUTsT 475 AGCUGGCUUUUCC 476 AD-9536 1118 GAAUAATsT 1100- uuAuucGGAAAAGccAGcuTsT 477 AGCUGGCUUUUCC 478 AD-9662 1118 GAAuAATsT 1154- CCCUGGCGGGUGGGUACAGTsT 479 CUGUACCCACCCG 480 AD-9584 1172 CCAGGGTsT 1154- cccuGGcGGGuGGGuAcAGTsT 481 CUGUACCCACCCG 482 AD-9710 1172 CcAGGGTsT 1155- CCUGGCGGGUGGGUACAGCTT 483 GCUGUACCCACCC 484 AD-15323 1173 GCCAGGTT 1157- UGGCGGGUGGGUACAGCCGTsT 485 CGGCUGUACCCAC 486 AD-9551 1175 CCGCCATsT 1157- uGGcGGGuGGGuAcAGccGTsT 487 CGGCUGuACCcAC 488 AD-9677 1175 CCGCcATsT 1158- GGCGGGUGGGUACAGCCGCTT 489 GCGGCUGUACCCA 490 AD-15230 1176 CCCGCCTT 1162- GGUGGGUACAGCCGCGUCCTT 491 GGACGCGGCUGUA 492 AD-15231 1180 CCCACCTT 1164- UGGGUACAGCCGCGUCCUCTT 493 GAGGACGCGGCUG 494 AD-15285 1182 UACCCATT 1172- GCCGCGUCCUCAACGCCGCTT 495 GCGGCGUUGAGGA 496 AD-15396 1190 CGCGGCTT 1173- CCGCGUCCUCAACGCCGCCTT 497 GGCGGCGUUGAGG 498 AD-15397 1191 ACGCGGTT 1216- GUCGUGCUGGUCACCGCUGTsT 499 CAGCGGUGACCAG 500 AD-9600 1234 CACGACTsT 1216- GucGuGcuGGucAccGcuGTsT 501 cAGCGGUGACcAG 502 AD-9726 1234 cACGACTsT 1217- UCGUGCUGGUCACCGCUGCTsT 503 GCAGCGGUGACCA 504 AD-9606 1235 GCACGATsT 1217- ucGuGcuGGucAccGcuGcTsT 505 GcAGCGGUGACcA 506 AD-9732 1235 GcACGATsT 1223- UGGUCACCGCUGCCGGCAATsT 507 UUGCCGGCAGCGG 508 AD-9633 1241 UGACCATsT 1223- uGGucAccGcuGccGGcAATsT 509 UUGCCGGcAGCGG 510 AD-9759 1241 UGACcATsT 1224- GGUCACCGCUGCCGGCAACTsT 511 GUUGCCGGCAGCG 512 AD-9588 1242 GUGACCTsT 1224- GGucAccGcuGccGGcAAcTsT 513 GUUGCCGGcAGCG 514 AD-9714 1242 GUGACCTsT 1227- CACCGCUGCCGGCAACUUCTsT 515 GAAGUUGCCGGCA 516 AD-9589 1245 GCGGUGTsT 1227- cAccGcuGccGGcAAcuucTsT 517 GAAGUUGCCGGcA 518 AD-9715 1245 GCGGUGTsT 1229- CCGCUGCCGGCAACUUCCGTsT 519 CGGAAGUUGCCGG 520 AD-9575 1247 CAGCGGTsT 1229- ccGcuGccGGcAAcuuccGTsT 521 CGGAAGUUGCCGG 522 AD-9701 1247 cAGCGGTsT 1230- CGCUGCCGGCAACUUCCGGTsT 523 CCGGAAGUUGCCG 524 AD-9563 1248 GCAGCGTsT 1230- cGcuGccGGcAAcuuccGGTsT 525 CCGGAAGUUGCCG 526 AD-9689 1248 GcAGCGTsT 1231- GCUGCCGGCAACUUCCGGGTsT 527 CCCGGAAGUUGCC 528 AD-9594 1249 GGCAGCTsT 1231- GcuGccGGcAAcuuccGGGTsT 529 CCCGGAAGUUGCC 530 AD-9720 1249 GGcAGCTsT 1236- CGGCAACUUCCGGGACGAUTsT 531 AUCGUCCCGGAAG 532 AD-9585 1254 UUGCCGTsT 1236- cGGcAAcuuccGGGAcGAuTsT 533 AUCGUCCCGGAAG 534 AD-9711 1254 UUGCCGTsT 1237- GGCAACUUCCGGGACGAUGTsT 535 CAUCGUCCCGGAA 536 AD-9614 1255 GUUGCCTsT 1237- GGcAAcuuccGGGAcGAuGTsT 537 cAUCGUCCCGGAA 538 AD-9740 1255 GUUGCCTsT 1243- UUCCGGGACGAUGCCUGCCTsT 539 GGCAGGCAUCGUC 540 AD-9615 1261 CCGGAATsT 1243- uuccGGGAcGAuGccuGccTsT 541 GGcAGGcAUCGUC 542 AD-9741 1261 CCGGAATsT 1248- GGACGAUGCCUGCCUCUACTsT 543 GUAGAGGCAGGCA 544 AD-9534 1266 UCGUCCTsT 1248- GGACGAUGCCUGCCUCUACTsT 545 GUAGAGGCAGGCA 546 AD-9534 1266 UCGUCCTsT 1248- GGAcGAuGccuGccucuAcTsT 547 GuAGAGGcAGGcA 548 AD-9660 1266 UCGUCCTsT 1279- GCUCCCGAGGUCAUCACAGTT 549 CUGUGAUGACCUC 550 AD-15324 1297 GGGAGCTT 1280- CUCCCGAGGUCAUCACAGUTT 551 ACUGUGAUGACCU 552 AD-15232 1298 CGGGAGTT 1281- UCCCGAGGUCAUCACAGUUTT 553 AACUGUGAUGACC 554 AD-15233 1299 UCGGGATT 1314- CCAAGACCAGCCGGUGACCTT 555 GGUCACCGGCUGG 556 AD-15234 1332 UCUUGGTT 1315- CAAGACCAGCCGGUGACCCTT 557 GGGUCACCGGCUG 558 AD-15286 1333 GUCUUGTT 1348- ACCAACUUUGGCCGCUGUGTsT 559 CACAGCGGCCAAA 560 AD-9590 1366 GUUGGUTsT 1348- AccAAcuuuGGccGcuGuGTsT 561 cAcAGCGGCcAAA 562 AD-9716 1366 GUUGGUTsT 1350- CAACUUUGGCCGCUGUGUGTsT 563 CACACAGCGGCCA 564 AD-9632 1368 AAGUUGTsT 1350- cAAcuuuGGccGcuGuGuGTsT 565 cAcAcAGCGGCcA 566 AD-9758 1368 AAGUUGTsT 1360- CGCUGUGUGGACCUCUUUGTsT 567 CAAAGAGGUCCAC 568 AD-9567 1378 ACAGCGTsT 1360- cGcuGuGuGGAccucuuuGTsT 569 cAAAGAGGUCcAc 570 AD-9693 1378 AcAGCGTsT 1390- GACAUCAUUGGUGCCUCCATsT 571 UGGAGGCACCAAU 572 AD-9586 1408 GAUGUCTsT 1390- GAcAucAuuGGuGccuccATsT 573 UGGAGGcACcAAU 574 AD-9712 1408 GAUGUCTsT 1394- UCAUUGGUGCCUCCAGCGATsT 575 UCGCUGGAGGCAC 576 AD-9564 1412 CAAUGATsT 1394- ucAuuGGuGccuccAGcGATsT 577 UCGCUGGAGGcAC 578 AD-9690 1412 cAAUGATsT 1417- AGCACCUGCUUUGUGUCACTsT 579 GUGACACAAAGCA 580 AD-9616 1435 GGUGCUTsT 1417- AGcAccuGcuuuGuGucAcTsT 581 GUGAcAcAAAGcA 582 AD-9742 1435 GGUGCUTsT 1433- CACAGAGUGGGACAUCACATT 583 UGUGAUGUCCCAC 584 AD-15398 1451 UCUGUGTT 1486- AUGCUGUCUGCCGAGCCGGTsT 585 CCGGCUCGGCAGA 586 AD-9617 1504 CAGCAUTsT 1486- AuGcuGucuGccGAGccGGTsT 587 CCGGCUCGGcAGA 588 AD-9743 1504 cAGcAUTsT 1491- GUCUGCCGAGCCGGAGCUCTsT 589 GAGCUCCGGCUCG 590 AD-9635 1509 GCAGACTsT 1491- GucuGccGAGccGGAGcucTsT 591 GAGCUCCGGCUCG 592 AD-9761 1509 GcAGACTsT 1521- GUUGAGGCAGAGACUGAUCTsT 593 GAUCAGUCUCUGC 594 AD-9568 1539 CUCAACTsT 1521- GuuGAGGcAGAGAcuGAucTsT 595 GAUcAGUCUCUGC 596 AD-9694 1539 CUcAACTsT 1527- GCAGAGACUGAUCCACUUCTsT 597 GAAGUGGAUCAGU 598 AD-9576 1545 CUCUGCTsT 1527- GcAGAGAcuGAuccAcuucTsT 599 GAAGUGGAUcAGU 600 AD-9702 1545 CUCUGCTsT 1529- AGAGACUGAUCCACUUCUCTsT 601 GAGAAGUGGAUCA 602 AD-9627 1547 GUCUCUTsT 1529- AGAGAcuGAuccAcuucucTsT 603 GAGAAGUGGAUcA 604 AD-9753 1547 GUCUCUTsT 1543- UUCUCUGCCAAAGAUGUCATsT 605 UGACAUCUUUGGC 606 AD-9628 1561 AGAGAATsT 1543- uucucuGccAAAGAuGucATsT 607 UGAcAUCUUUGGc 608 AD-9754 1561 AGAGAATsT 1545- CUCUGCCAAAGAUGUCAUCTsT 609 GAUGACAUCUUUG 610 AD-9631 1563 GCAGAGTsT 1545- cucuGccAAAGAuGucAucTsT 611 GAUGAcAUCUUUG 612 AD-9757 1563 GcAGAGTsT 1580- CUGAGGACCAGCGGGUACUTsT 613 AGUACCCGCUGGU 614 AD-9595 1598 CCUCAGTsT 1580- cuGAGGAccAGcGGGuAcuTsT 615 AGuACCCGCUGGU 616 AD-9721 1598 CCUcAGTsT 1581- UGAGGACCAGCGGGUACUGTsT 617 CAGUACCCGCUGG 618 AD-9544 1599 UCCUCATsT 1581- uGAGGAccAGcGGGuAcuGTsT 619 cAGuACCCGCUGG 620 AD-9670 1599 UCCUcATsT 1666- ACUGUAUGGUCAGCACACUTT 621 AGUGUGCUGACCA 622 AD-15235 1684 UACAGUTT 1668- UGUAUGGUCAGCACACUCGTT 623 CGAGUGUGCUGAC 624 AD-15236 1686 CAUACATT 1669- GUAUGGUCAGCACACUCGGTT 625 CCGAGUGUGCUGA 626 AD-15168 1687 CCAUACTT 1697- GGAUGGCCACAGCCGUCGCTT 627 GCGACGGCUGUGG 628 AD-15174 1715 CCAUCCTT 1698- GAUGGCCACAGCCGUCGCCTT 629 GGCGACGGCUGUG 630 AD-15325 1716 GCCAUCTT 1806- CAAGCUGGUCUGCCGGGCCTT 631 GGCCCGGCAGACC 632 AD-15326 1824 AGCUUGTT 1815- CUGCCGGGCCCACAACGCUTsT 633 AGCGUUGUGGGCC 634 AD-9570 1833 CGGCAGTsT 1815- cuGccGGGcccAcAAcGcuTsT 635 AGCGUUGUGGGCC 636 AD-9696 1833 CGGcAGTsT 1816- UGCCGGGCCCACAACGCUUTsT 637 AAGCGUUGUGGGC 638 AD-9566 1834 CCGGCATsT 1816- uGccGGGcccAcAAcGcuuTsT 639 AAGCGUUGUGGGC 640 AD-9692 1834 CCGGcATsT 1818- CCGGGCCCACAACGCUUUUTsT 641 AAAAGCGUUGUGG 642 AD-9532 1836 GCCCGGTsT 1818- ccGGGcccAcAAcGcuuuuTsT 643 AAAAGCGUUGUGG 644 AD-9658 1836 GCCCGGTsT 1820- GGGCCCACAACGCUUUUGGTsT 645 CCAAAAGCGUUGU 646 AD-9549 1838 GGGCCCTsT 1820- GGGcccAcAAcGcuuuuGGTsT 647 CcAAAAGCGUUGU 648 AD-9675 1838 GGGCCCTsT 1840- GGUGAGGGUGUCUACGCCATsT 649 UGGCGUAGACACC 650 AD-9541 1858 CUCACCTsT 1840- GGuGAGGGuGucuAcGccATsT 651 UGGCGUAGACACC 652 AD-9667 1858 CUcACCTsT 1843- GAGGGUGUCUACGCCAUUGTsT 653 CAAUGGCGUAGAC 654 AD-9550 1861 ACCCUCTsT 1843- GAGGGuGucuAcGccAuuGTsT 655 cAAUGGCGuAGAc 656 AD-9676 1861 ACCCUCTsT 1861- GCCAGGUGCUGCCUGCUACTsT 657 GUAGCAGGCAGCA 658 AD-9571 1879 CCUGGCTsT 1861- GccAGGuGcuGccuGcuAcTsT 659 GuAGcAGGcAGcA 660 AD-9697 1879 CCUGGCTsT 1862- CCAGGUGCUGCCUGCUACCTsT 661 GGUAGCAGGCAGC 662 AD-9572 1880 ACCUGGTsT 1862- ccAGGuGcuGccuGcuAccTsT 663 GGuAGcAGGcAGc 664 AD-9698 1880 ACCUGGTsT 2008- ACCCACAAGCCGCCUGUGCTT 665 GCACAGGCGGCUU 666 AD-15327 2026 GUGGGUTT 2023- GUGCUGAGGCCACGAGGUCTsT 667 GACCUCGUGGCCU 668 AD-9639 2041 CAGCACTsT 2023- GuGcuGAGGccAcGAGGucTsT 669 GACCUCGUGGCCU 670 AD-9765 2041 cAGcACTsT 2024- UGCUGAGGCCACGAGGUCATsT 671 UGACCUCGUGGCC 672 AD-9518 2042 UCAGCATsT 2024- UGCUGAGGCCACGAGGUCATsT 673 UGACCUCGUGGCC 674 AD-9518 2042 UCAGCATsT 2024- uGcuGAGGccAcGAGGucATsT 675 UGACCUCGUGGCC 676 AD-9644 2042 UcAGcATsT 2024- UfgCfuGfaGfgCfcAfcGfaG 677 P*uGfaCfcUfcG 678 AD-14672 2042 fgUfcAfTsT fuGfgCfcUfcAf gCfaTsT 2024- UfGCfUfGAGGCfCfACfGAGG 679 UfGACfCfUfCfG 680 AD-14682 2042 UfCfATsT UfGGCfCfUfCfA GCfATsT 2024- UgCuGaGgCcAcGaGgUcATsT 681 P*uGfaCfcUfcG 682 AD-14692 2042 fuGfgCfcUfcAf gCfaTsT 2024- UgCuGaGgCcAcGaGgUcATsT 683 UfGACfCfUfCfG 684 AD-14702 2042 UfGGCfCfUfCfA GCfATsT 2024- UfgCfuGfaGfgCfcAfcGfaG 685 UGACCucGUggCC 686 AD-14712 2042 fgUfcAfTsT UCAgcaTsT 2024- UfGCfUfGAGGCfCfACfGAGG 687 UGACCucGUggCC 688 AD-14722 2042 UfCfATsT UCAgcaTsT 2024- UgCuGaGgCcAcGaGgUcATsT 689 UGACCucGUggCC 690 AD-14732 2042 UCAgcaTsT 2024- GfuGfgUfcAfgCfgGfcCfgG 691 P*cAfuCfcCfgG 692 AD-15078 2042 fgAfuGfTsT fcCfgCfuGfaCf cAfcTsT 2024- GUfGGUfCfAGCfGGCfCfGGG 693 CfAUfCfCfCfGG 694 AD-15088 2042 AUfGTsT CfCfGCfUfGACf CfACfTsT 2024- GuGgUcAgCgGcCgGgAuGTsT 695 P*cAfuCfcCfgG 696 AD-15098 2042 fcCfgCfuGfaCf cAfcTsT 2024- GuGgUcAgCgGcCgGgAuGTsT 697 CfAUfCfCfCfGG 698 AD-15108 2042 CfCfGCfUfGACf CfACfTsT 2024- GfuGfgUfcAfgCfgGfcCfgG 699 CAUCCcgGCcgCU 700 AD-15118 2042 fgAfuGfTsT GACcacTsT 2024- GUfGGUfCfAGCfGGCfCfGGG 701 CAUCCcgGCcgCU 702 AD-15128 2042 AUfGTsT GACcacTsT 2024- GuGgUcAgCgGcCgGgAuGTsT 703 CAUCCcgGCcgCU 704 AD-15138 2042 GACcacTsT 2030- GGCCACGAGGUCAGCCCAATT 705 UUGGGCUGACCUC 706 AD-15237 2048 GUGGCCTT 2035- CGAGGUCAGCCCAACCAGUTT 707 ACUGGUUGGGCUG 708 AD-15287 2053 ACCUCGTT 2039- GUCAGCCCAACCAGUGCGUTT 709 ACGCACUGGUUGG 710 AD-15238 2057 GCUGACTT 2041- CAGCCCAACCAGUGCGUGGTT 711 CCACGCACUGGUU 712 AD-15328 2059 GGGCUGTT 2062- CACAGGGAGGCCAGCAUCCTT 713 GGAUGCUGGCCUC 714 AD-15399 2080 CCUGUGTT 2072- CCAGCAUCCACGCUUCCUGTsT 715 CAGGAAGCGUGGA 716 AD-9582 2090 UGCUGGTsT 2072- ccAGcAuccAcGcuuccuGTsT 717 cAGGAAGCGUGGA 718 AD-9708 2090 UGCUGGTsT 2118- AGUCAAGGAGCAUGGAAUCTsT 719 GAUUCCAUGCUCC 720 AD-9545 2136 UUGACUTsT 2118- AGucAAGGAGcAuGGAAucTsT 721 GAUUCcAUGCUCC 722 AD-9671 2136 UUGACUTsT 2118- AfgUfcAfaGfgAfgCfaUfgG 723 P*gAfuUfcCfaU 724 AD-14674 2136 faAfuCfTsT fgCfuCfcUfuGf aCfuTsT 2118- AGUfCfAAGGAGCfAUfGGAAU 725 GAUfUfCfCfAUf 726 AD-14684 2136 fCfTsT GCfUfCfCfUfUf GACfUfTsT 2118- AgUcAaGgAgCaUgGaAuCTsT 727 P*gAfuUfcCfaU 728 AD-14694 2136 fgCfuCfcUfuGf aCfuTsT 2118- AgUcAaGgAgCaUgGaAuCTsT 729 GAUfUfCfCfAUf 730 AD-14704 2136 GCfUfCfCfUfUf GACfUfTsT 2118- AfgUfcAfaGfgAfgCfaUfgG 731 GAUUCcaUGcuCC 732 AD-14714 2136 faAfuCfTsT UUGacuTsT 2118- AGUfCfAAGGAGCfAUfGGAAU 733 GAUUCcaUGcuCC 734 AD-14724 2136 fCfTsT UUGacuTsT 2118- AgUcAaGgAgCaUgGaAuCTsT 735 GAUUCcaUGcuCC 736 AD-14734 2136 UUGacuTsT 2118- GfcGfgCfaCfcCfuCfaUfaG 737 P*aGfgCfcUfaU 738 AD-15080 2136 fgCfcUfTsT fgAfgGfgUfgCf cGfcTsT 2118- GCfGGCfACfCfCfUfCfAUfA 739 AGGCfCfUfAUfG 740 AD-15090 2136 GGCfCfUfTsT AGGGUfGCfCfGC fTsT 2118- GcGgCaCcCuCaUaGgCcUTsT 741 P*aGfgCfcUfaU 742 AD-15100 2136 fgAfgGfgUfgCf cGfcTsT 2118- GcGgCaCcCuCaUaGgCcUTsT 743 AGGCfCfUfAUfG 744 AD-15110 2136 AGGGUfGCfCfGC fTsT 2118- GfcGfgCfaCfcCfuCfaUfaG 745 AGGCCuaUGagGG 746 AD-15120 2136 fgCfcUfTsT UGCcgcTsT 2118- GCfGGCfACfCfCfUfCfAUfA 747 AGGCCuaUGagGG 748 AD-15130 2136 GGCfCfUfTsT UGCcgcTsT 2118- GcGgCaCcCuCaUaGgCcUTsT 749 AGGCCuaUGagGG 750 AD-15140 2136 UGCcgcTsT 2122- AAGGAGCAUGGAAUCCCGGTsT 751 CCGGGAUUCCAUG 752 AD-9522 2140 CUCCUUTsT 2122- AAGGAGcAuGGAAucccGGTsT 753 CCGGGAUUCcAUG 754 AD-9648 2140 CUCCUUTsT 2123- AGGAGCAUGGAAUCCCGGCTsT 755 GCCGGGAUUCCAU 756 AD-9552 2141 GCUCCUTsT 2123- AGGAGcAuGGAAucccGGcTsT 757 GCCGGGAUUCcAU 758 AD-9678 2141 GCUCCUTsT 2125- GAGCAUGGAAUCCCGGCCCTsT 759 GGGCCGGGAUUCC 760 AD-9618 2143 AUGCUCTsT 2125- GAGcAuGGAAucccGGcccTsT 761 GGGCCGGGAUUCc 762 AD-9744 2143 AUGCUCTsT 2230- GCCUACGCCGUAGACAACATT 763 UGUUGUCUACGGC 764 AD-15239 2248 GUAGGCTT 2231- CCUACGCCGUAGACAACACTT 765 GUGUUGUCUACGG 766 AD-15212 2249 CGUAGGTT 2232- CUACGCCGUAGACAACACGTT 767 CGUGUUGUCUACG 768 AD-15240 2250 GCGUAGTT 2233- UACGCCGUAGACAACACGUTT 769 ACGUGUUGUCUAC 770 AD-15177 2251 GGCGUATT 2235- CGCCGUAGACAACACGUGUTT 771 ACACGUGUUGUCU 772 AD-15179 2253 ACGGCGTT 2236- GCCGUAGACAACACGUGUGTT 773 CACACGUGUUGUC 774 AD-15180 2254 UACGGCTT 2237- CCGUAGACAACACGUGUGUTT 775 ACACACGUGUUGU 776 AD-15241 2255 CUACGGTT 2238- CGUAGACAACACGUGUGUATT 777 UACACACGUGUUG 778 AD-15268 2256 UCUACGTT 2240- UAGACAACACGUGUGUAGUTT 779 ACUACACACGUGU 780 AD-15242 2258 UGUCUATT 2241- AGACAACACGUGUGUAGUCTT 781 GACUACACACGUG 782 AD-15216 2259 UUGUCUTT 2242- GACAACACGUGUGUAGUCATT 783 UGACUACACACGU 784 AD-15176 2260 GUUGUCTT 2243- ACAACACGUGUGUAGUCAGTT 785 CUGACUACACACG 786 AD-15181 2261 UGUUGUTT 2244- CAACACGUGUGUAGUCAGGTT 787 CCUGACUACACAC 788 AD-15243 2262 GUGUUGTT 2247- CACGUGUGUAGUCAGGAGCTT 789 GCUCCUGACUACA 790 AD-15182 2265 CACGUGTT 2248- ACGUGUGUAGUCAGGAGCCTT 791 GGCUCCUGACUAC 792 AD-15244 2266 ACACGUTT 2249- CGUGUGUAGUCAGGAGCCGTT 793 CGGCUCCUGACUA 794 AD-15387 2267 CACACGTT 2251- UGUGUAGUCAGGAGCCGGGTT 795 CCCGGCUCCUGAC 796 AD-15245 2269 UACACATT 2257- GUCAGGAGCCGGGACGUCATsT 797 UGACGUCCCGGCU 798 AD-9555 2275 CCUGACTsT 2257- GucAGGAGccGGGAcGucATsT 799 UGACGUCCCGGCU 800 AD-9681 2275 CCUGACTsT 2258- UCAGGAGCCGGGACGUCAGTsT 801 CUGACGUCCCGGC 802 AD-9619 2276 UCCUGATsT 2258- ucAGGAGccGGGAcGucAGTsT 803 CUGACGUCCCGGC 804 AD-9745 2276 UCCUGATsT 2259- CAGGAGCCGGGACGUCAGCTsT 805 GCUGACGUCCCGG 806 AD-9620 2277 CUCCUGTsT 2259- cAGGAGccGGGAcGucAGcTsT 807 GCUGACGUCCCGG 808 AD-9746 2277 CUCCUGTsT 2263- AGCCGGGACGUCAGCACUATT 809 UAGUGCUGACGUC 810 AD-15288 2281 CCGGCUTT 2265- CCGGGACGUCAGCACUACATT 811 UGUAGUGCUGACG 812 AD-15246 2283 UCCCGGTT 2303- CCGUGACAGCCGUUGCCAUTT 813 AUGGCAACGGCUG 814 AD-15289 2321 UCACGGTT 2317- GCCAUCUGCUGCCGGAGCCTsT 815 GGCUCCGGCAGCA 816 AD-9324 2335 GAUGGCTsT 2375- CCCAUCCCAGGAUGGGUGUTT 817 ACACCCAUCCUGG 818 AD-15329 2393 GAUGGGTT 2377- CAUCCCAGGAUGGGUGUCUTT 819 AGACACCCAUCCU 820 AD-15330 2395 GGGAUGTT 2420- AGCUUUAAAAUGGUUCCGATT 821 UCGGAACCAUUUU 822 AD-15169 2438 AAAGCUTT 2421- GCUUUAAAAUGGUUCCGACTT 823 GUCGGAACCAUUU 824 AD-15201 2439 UAAAGCTT 2422- CUUUAAAAUGGUUCCGACUTT 825 AGUCGGAACCAUU 826 AD-15331 2440 UUAAAGTT 2423- UUUAAAAUGGUUCCGACUUTT 827 AAGUCGGAACCAU 828 AD-15190 2441 UUUAAATT 2424- UUAAAAUGGUUCCGACUUGTT 829 CAAGUCGGAACCA 830 AD-15247 2442 UUUUAATT 2425- UAAAAUGGUUCCGACUUGUTT 831 ACAAGUCGGAACC 832 AD-15248 2443 AUUUUATT 2426- AAAAUGGUUCCGACUUGUCTT 833 GACAAGUCGGAAC 834 AD-15175 2444 CAUUUUTT 2427- AAAUGGUUCCGACUUGUCCTT 835 GGACAAGUCGGAA 836 AD-15249 2445 CCAUUUTT 2428- AAUGGUUCCGACUUGUCCCTT 837 GGGACAAGUCGGA 838 AD-15250 2446 ACCAUUTT 2431- GGUUCCGACUUGUCCCUCUTT 839 AGAGGGACAAGUC 840 AD-15400 2449 GGAACCTT 2457- CUCCAUGGCCUGGCACGAGTT 841 CUCGUGCCAGGCC 842 AD-15332 2475 AUGGAGTT 2459- CCAUGGCCUGGCACGAGGGTT 843 CCCUCGUGCCAGG 844 AD-15388 2477 CCAUGGTT 2545- GAACUCACUCACUCUGGGUTT 845 ACCCAGAGUGAGU 846 AD-15333 2563 GAGUUCTT 2549- UCACUCACUCUGGGUGCCUTT 847 AGGCACCCAGAGU 848 AD-15334 2567 GAGUGATT 2616- UUUCACCAUUCAAACAGGUTT 849 ACCUGUUUGAAUG 850 AD-15335 2634 GUGAAATT 2622- CAUUCAAACAGGUCGAGCUTT 851 AGCUCGACCUGUU 852 AD-15183 2640 UGAAUGTT 2623- AUUCAAACAGGUCGAGCUGTT 853 CAGCUCGACCUGU 854 AD-15202 2641 UUGAAUTT 2624- UUCAAACAGGUCGAGCUGUTT 855 ACAGCUCGACCUG 856 AD-15203 2642 UUUGAATT 2625- UCAAACAGGUCGAGCUGUGTT 857 CACAGCUCGACCU 858 AD-15272 2643 GUUUGATT 2626- CAAACAGGUCGAGCUGUGCTT 859 GCACAGCUCGACC 860 AD-15217 2644 UGUUUGTT 2627- AAACAGGUCGAGCUGUGCUTT 861 AGCACAGCUCGAC 862 AD-15290 2645 CUGUUUTT 2628- AACAGGUCGAGCUGUGCUCTT 863 GAGCACAGCUCGA 864 AD-15218 2646 CCUGUUTT 2630- CAGGUCGAGCUGUGCUCGGTT 865 CCGAGCACAGCUC 866 AD-15389 2648 GACCUGTT 2631- AGGUCGAGCUGUGCUCGGGTT 867 CCCGAGCACAGCU 868 AD-15336 2649 CGACCUTT 2633- GUCGAGCUGUGCUCGGGUGTT 869 CACCCGAGCACAG 870 AD-15337 2651 CUCGACTT 2634- UCGAGCUGUGCUCGGGUGCTT 871 GCACCCGAGCACA 872 AD-15191 2652 GCUCGATT 2657- AGCUGCUCCCAAUGUGCCGTT 873 CGGCACAUUGGGA 874 AD-15390 2675 GCAGCUTT 2658- GCUGCUCCCAAUGUGCCGATT 875 UCGGCACAUUGGG 876 AD-15338 2676 AGCAGCTT 2660- UGCUCCCAAUGUGCCGAUGTT 877 CAUCGGCACAUUG 878 AD-15204 2678 GGAGCATT 2663- UCCCAAUGUGCCGAUGUCCTT 879 GGACAUCGGCACA 880 AD-15251 2681 UUGGGATT 2665- CCAAUGUGCCGAUGUCCGUTT 881 ACGGACAUCGGCA 882 AD-15205 2683 CAUUGGTT 2666- CAAUGUGCCGAUGUCCGUGTT 883 CACGGACAUCGGC 884 AD-15171 2684 ACAUUGTT 2667- AAUGUGCCGAUGUCCGUGGTT 885 CCACGGACAUCGG 886 AD-15252 2685 CACAUUTT 2673- CCGAUGUCCGUGGGCAGAATT 887 UUCUGCCCACGGA 888 AD-15339 2691 CAUCGGTT 2675- GAUGUCCGUGGGCAGAAUGTT 889 CAUUCUGCCCACG 890 AD-15253 2693 GACAUCTT 2678- GUCCGUGGGCAGAAUGACUTT 891 AGUCAUUCUGCCC 892 AD-15340 2696 ACGGACTT 2679- UCCGUGGGCAGAAUGACUUTT 893 AAGUCAUUCUGCC 894 AD-15291 2697 CACGGATT 2683- UGGGCAGAAUGACUUUUAUTT 895 AUAAAAGUCAUUC 896 AD-15341 2701 UGCCCATT 2694- ACUUUUAUUGAGCUCUUGUTT 897 ACAAGAGCUCAAU 898 AD-15401 2712 AAAAGUTT 2700- AUUGAGCUCUUGUUCCGUGTT 899 CACGGAACAAGAG 900 AD-15342 2718 CUCAAUTT 2704- AGCUCUUGUUCCGUGCCAGTT 901 CUGGCACGGAACA 902 AD-15343 2722 AGAGCUTT 2705- GCUCUUGUUCCGUGCCAGGTT 903 CCUGGCACGGAAC 904 AD-15292 2723 AAGAGCTT 2710- UGUUCCGUGCCAGGCAUUCTT 905 GAAUGCCUGGCAC 906 AD-15344 2728 GGAACATT 2711- GUUCCGUGCCAGGCAUUCATT 907 UGAAUGCCUGGCA 908 AD-15254 2729 CGGAACTT 2712- UUCCGUGCCAGGCAUUCAATT 909 UUGAAUGCCUGGC 910 AD-15345 2730 ACGGAATT 2715- CGUGCCAGGCAUUCAAUCCTT 911 GGAUUGAAUGCCU 912 AD-15206 2733 GGCACGTT 2716- GUGCCAGGCAUUCAAUCCUTT 913 AGGAUUGAAUGCC 914 AD-15346 2734 UGGCACTT 2728- CAAUCCUCAGGUCUCCACCTT 915 GGUGGAGACCUGA 916 AD-15347 2746 GGAUUGTT 2743- CACCAAGGAGGCAGGAUUCTsT 917 GAAUCCUGCCUCC 918 AD-9577 2761 UUGGUGTsT 2743- cAccAAGGAGGcAGGAuucTsT 919 GAAUCCUGCCUCC 920 AD-9703 2761 UUGGUGTsT 2743- CfaCfcAfaGfgAfgGfcAfgG 921 P*gAfaUfcCfuG 922 AD-14678 2761 faUfuCfTsT fcCfuCfcUfuGf gUfgTsT 2743- CfACfCfAAGGAGGCfAGGAUf 923 GAAUfCfCfUfGC 924 AD-14688 2761 UfCfTsT fCfUfCfCfUfUf GGUfGTsT 2743- CaCcAaGgAgGcAgGaUuCTsT 925 P*gAfaUfcCfuG 926 AD-14698 2761 fcCfuCfcUfuGf gUfgTsT 2743- CaCcAaGgAgGcAgGaUuCTsT 927 GAAUfCfCfUfGC 928 AD-14708 2761 fCfUfCfCfUfUf GGUfGTsT 2743- CfaCfcAfaGfgAfgGfcAfgG 929 GAAUCcuGCcuCC 930 AD-14718 2761 faUfuCfTsT UUGgugTsT 2743- CfACfCfAAGGAGGCfAGGAUf 931 GAAUCcuGCcuCC 932 AD-14728 2761 UfCfTsT UUGgugTsT 2743- CaCcAaGgAgGcAgGaUuCTsT 933 GAAUCcuGCcuCC 934 AD-14738 2761 UUGgugTsT 2743- GfgCfcUfgGfaGfuUfuAfuU 935 P*uCfcGfaAfuA 936 AD-15084 2761 fcGfgAfTsT faAfcUfcCfaGf gCfcTsT 2743- GGCfCfUfGGAGUfUfUfAUfU 937 UfCfCfGAAUfAA 938 AD-15094 2761 fCfGGATsT ACfUfCfCfAGGC fCfTsT 2743- GgCcUgGaGuUuAuUcGgATsT 939 P*uCfcGfaAfuA 940 AD-15104 2761 faAfcUfcCfaGf gCfcTsT 2743- GgCcUgGaGuUuAuUcGgATsT 941 UfCfCfGAAUfAA 942 AD-15114 2761 ACfUfCfCfAGGC fCfTsT 2743- GfgCfcUfgGfaGfuUfuAfuU 943 UCCGAauAAacUC 944 AD-15124 2761 fcGfgAfTsT CAGgccTsT 2743- GGCfCfUfGGAGUfUfUfAUfU 945 UCCGAauAAacUC 946 AD-15134 2761 fCfGGATsT CAGgccTsT 2743- GgCcUgGaGuUuAuUcGgATsT 947 UCCGAauAAacUC 948 AD-15144 2761 CAGgccTsT 2753- GCAGGAUUCUUCCCAUGGATT 949 UCCAUGGGAAGAA 950 AD-15391 2771 UCCUGCTT 2794- UGCAGGGACAAACAUCGUUTT 951 AACGAUGUUUGUC 952 AD-15348 2812 CCUGCATT 2795- GCAGGGACAAACAUCGUUGTT 953 CAACGAUGUUUGU 954 AD-15349 2813 CCCUGCTT 2797- AGGGACAAACAUCGUUGGGTT 955 CCCAACGAUGUUU 956 AD-15170 2815 GUCCCUTT 2841- CCCUCAUCUCCAGCUAACUTT 957 AGUUAGCUGGAGA 958 AD-15350 2859 UGAGGGTT 2845- CAUCUCCAGCUAACUGUGGTT 959 CCACAGUUAGCUG 960 AD-15402 2863 GAGAUGTT 2878- GCUCCCUGAUUAAUGGAGGTT 961 CCUCCAUUAAUCA 962 AD-15293 2896 GGGAGCTT 2881- CCCUGAUUAAUGGAGGCUUTT 963 AAGCCUCCAUUAA 964 AD-15351 2899 UCAGGGTT 2882- CCUGAUUAAUGGAGGCUUATT 965 UAAGCCUCCAUUA 966 AD-15403 2900 AUCAGGTT 2884- UGAUUAAUGGAGGCUUAGCTT 967 GCUAAGCCUCCAU 968 AD-15404 2902 UAAUCATT 2885- GAUUAAUGGAGGCUUAGCUTT 969 AGCUAAGCCUCCA 970 AD-15207 2903 UUAAUCTT 2886- AUUAAUGGAGGCUUAGCUUTT 971 AAGCUAAGCCUCC 972 AD-15352 2904 AUUAAUTT 2887- UUAAUGGAGGCUUAGCUUUTT 973 AAAGCUAAGCCUC 974 AD-15255 2905 CAUUAATT 2903- UUUCUGGAUGGCAUCUAGCTsT 975 GCUAGAUGCCAUC 976 AD-9603 2921 CAGAAATsT 2903- uuucuGGAuGGcAucuAGcTsT 977 GCuAGAUGCcAUC 978 AD-9729 2921 cAGAAATsT 2904- UUCUGGAUGGCAUCUAGCCTsT 979 GGCUAGAUGCCAU 980 AD-9599 2922 CCAGAATsT 2904- uucuGGAuGGcAucuAGccTsT 981 GGCuAGAUGCcAU 982 AD-9725 2922 CcAGAATsT 2905- UCUGGAUGGCAUCUAGCCATsT 983 UGGCUAGAUGCCA 984 AD-9621 2923 UCCAGATsT 2905- ucuGGAuGGcAucuAGccATsT 985 UGGCuAGAUGCcA 986 AD-9747 2923 UCcAGATsT 2925- AGGCUGGAGACAGGUGCGCTT 987 GCGCACCUGUCUC 988 AD-15405 2943 CAGCCUTT 2926- GGCUGGAGACAGGUGCGCCTT 989 GGCGCACCUGUCU 990 AD-15353 2944 CCAGCCTT 2927- GCUGGAGACAGGUGCGCCCTT 991 GGGCGCACCUGUC 992 AD-15354 2945 UCCAGCTT 2972- UUCCUGAGCCACCUUUACUTT 993 AGUAAAGGUGGCU 994 AD-15406 2990 CAGGAATT 2973- UCCUGAGCCACCUUUACUCTT 995 GAGUAAAGGUGGC 996 AD-15407 2991 UCAGGATT 2974- CCUGAGCCACCUUUACUCUTT 997 AGAGUAAAGGUGG 998 AD-15355 2992 CUCAGGTT 2976- UGAGCCACCUUUACUCUGCTT 999 GCAGAGUAAAGGU 1000 AD-15356 2994 GGCUCATT 2978- AGCCACCUUUACUCUGCUCTT 1001 GAGCAGAGUAAAG 1002 AD-15357 2996 GUGGCUTT 2981- CACCUUUACUCUGCUCUAUTT 1003 AUAGAGCAGAGUA 1004 AD-15269 2999 AAGGUGTT 2987- UACUCUGCUCUAUGCCAGGTsT 1005 CCUGGCAUAGAGC 1006 AD-9565 3005 AGAGUATsT 2987- uAcucuGcucuAuGccAGGTsT 1007 CCUGGcAuAGAGc 1008 AD-9691 3005 AGAGuATsT 2998- AUGCCAGGCUGUGCUAGCATT 1009 UGCUAGCACAGCC 1010 AD-15358 3016 UGGCAUTT 3003- AGGCUGUGCUAGCAACACCTT 1011 GGUGUUGCUAGCA 1012 AD-15359 3021 CAGCCUTT 3006- CUGUGCUAGCAACACCCAATT 1013 UUGGGUGUUGCUA 1014 AD-15360 3024 GCACAGTT 3010- GCUAGCAACACCCAAAGGUTT 1015 ACCUUUGGGUGUU 1016 AD-15219 3028 GCUAGCTT 3038- GGAGCCAUCACCUAGGACUTT 1017 AGUCCUAGGUGAU 1018 AD-15361 3056 GGCUCCTT 3046- CACCUAGGACUGACUCGGCTT 1019 GCCGAGUCAGUCC 1020 AD-15273 3064 UAGGUGTT 3051- AGGACUGACUCGGCAGUGUTT 1021 ACACUGCCGAGUC 1022 AD-15362 3069 AGUCCUTT 3052- GGACUGACUCGGCAGUGUGTT 1023 CACACUGCCGAGU 1024 AD-15192 3070 CAGUCCTT 3074- UGGUGCAUGCACUGUCUCATT 1025 UGAGACAGUGCAU 1026 AD-15256 3092 GCACCATT 3080- AUGCACUGUCUCAGCCAACTT 1027 GUUGGCUGAGACA 1028 AD-15363 3098 GUGCAUTT 3085- CUGUCUCAGCCAACCCGCUTT 1029 AGCGGGUUGGCUG 1030 AD-15364 3103 AGACAGTT 3089- CUCAGCCAACCCGCUCCACTsT 1031 GUGGAGCGGGUUG 1032 AD-9604 3107 GCUGAGTsT 3089- cucAGccAAcccGcuccAcTsT 1033 GUGGAGCGGGUUG 1034 AD-9730 3107 GCUGAGTsT 3093- GCCAACCCGCUCCACUACCTsT 1035 GGUAGUGGAGCGG 1036 AD-9527 3111 GUUGGCTsT 3093- GccAAcccGcuccAcuAccTsT 1037 GGuAGUGGAGCGG 1038 AD-9653 3111 GUUGGCTsT 3096- AACCCGCUCCACUACCCGGTT 1039 CCGGGUAGUGGAG 1040 AD-15365 3114 CGGGUUTT 3099- CCGCUCCACUACCCGGCAGTT 1041 CUGCCGGGUAGUG 1042 AD-15294 3117 GAGCGGTT 3107- CUACCCGGCAGGGUACACATT 1043 UGUGUACCCUGCC 1044 AD-15173 3125 GGGUAGTT 3108- UACCCGGCAGGGUACACAUTT 1045 AUGUGUACCCUGC 1046 AD-15366 3126 CGGGUATT 3109- ACCCGGCAGGGUACACAUUTT 1047 AAUGUGUACCCUG 1048 AD-15367 3127 CCGGGUTT 3110- CCCGGCAGGGUACACAUUCTT 1049 GAAUGUGUACCCU 1050 AD-15257 3128 GCCGGGTT 3112- CGGCAGGGUACACAUUCGCTT 1051 GCGAAUGUGUACC 1052 AD-15184 3130 CUGCCGTT 3114- GCAGGGUACACAUUCGCACTT 1053 GUGCGAAUGUGUA 1054 AD-15185 3132 CCCUGCTT 3115- CAGGGUACACAUUCGCACCTT 1055 GGUGCGAAUGUGU 1056 AD-15258 3133 ACCCUGTT 3116- AGGGUACACAUUCGCACCCTT 1057 GGGUGCGAAUGUG 1058 AD-15186 3134 UACCCUTT 3196- GGAACUGAGCCAGAAACGCTT 1059 GCGUUUCUGGCUC 1060 AD-15274 3214 AGUUCCTT 3197- GAACUGAGCCAGAAACGCATT 1061 UGCGUUUCUGGCU 1062 AD-15368 3215 CAGUUCTT 3198- AACUGAGCCAGAAACGCAGTT 1063 CUGCGUUUCUGGC 1064 AD-15369 3216 UCAGUUTT 3201- UGAGCCAGAAACGCAGAUUTT 1065 AAUCUGCGUUUCU 1066 AD-15370 3219 GGCUCATT 3207- AGAAACGCAGAUUGGGCUGTT 1067 CAGCCCAAUCUGC 1068 AD-15259 3225 GUUUCUTT 3210- AACGCAGAUUGGGCUGGCUTT 1069 AGCCAGCCCAAUC 1070 AD-15408 3228 UGCGUUTT 3233- AGCCAAGCCUCUUCUUACUTsT 1071 AGUAAGAAGAGGC 1072 AD-9597 3251 UUGGCUTsT 3233- AGccAAGccucuucuuAcuTsT 1073 AGuAAGAAGAGGC 1074 AD-9723 3251 UUGGCUTsT 3233- AfgCfcAfaGfcCfuCfuUfcU 1075 P*aGfuAfaGfaA 1076 AD-14680 3251 fuAfcUfTsT fgAfgGfcUfuGf gCfuTsT 3233- AGCfCfAAGCfCfUfCfUfUfC 1077 AGUfAAGAAGAGG 1078 AD-14690 3251 fUfUfACfUfTsT CfUfUfGGCfUfT sT 3233- AgCcAaGcCuCuUcUuAcUTsT 1079 P*aGfuAfaGfaA 1080 AD-14700 3251 fgAfgGfcUfuGf gCfuTsT 3233- AgCcAaGcCuCuUcUuAcUTsT 1081 AGUfAAGAAGAGG 1082 AD-14710 3251 CfUfUfGGCfUfT sT 3233- AfgCfcAfaGfcCfuCfuUfcU 1083 AGUAAgaAGagGC 1084 AD-14720 3251 fuAfcUfTsT UUGgcuTsT 3233- AGCfCfAAGCfCfUfCfUfUfC 1085 AGUAAgaAGagGC 1086 AD-14730 3251 fUfUfACfUfTsT UUGgcuTsT 3233- AgCcAaGcCuCuUcUuAcUTsT 1087 AGUAAgaAGagGC 1088 AD-14740 3251 UUGgcuTsT 3233- UfgGfuUfcCfcUfgAfgGfaC 1089 P*gCfuGfgUfcC 1090 AD-15086 3251 fcAfgCfTsT fuCfaGfgGfaAf cCfaTsT 3233- UfGGUfUfCfCfCfUfGAGGAC 1091 GCfUfGGUfCfCf 1092 AD-15096 3251 fCfAGCfTsT UfCfAGGGAACfC fATsT 3233- UgGuUcCcUgAgGaCcAgCTsT 1093 P*gCfuGfgUfcC 1094 AD-15106 3251 fuCfaGfgGfaAf cCfaTsT 3233- UgGuUcCcUgAgGaCcAgCTsT 1095 GCfUfGGUfCfCf 1096 AD-15116 3251 UfCfAGGGAACfC fATsT 3233- UfgGfuUfcCfcUfgAfgGfaC 1097 GCUGGucCUcaGG 1098 AD-15126 3251 fcAfgCfTsT GAAccaTsT 3233- UfGGUfUfCfCfCfUfGAGGAC 1099 GCUGGucCUcaGG 1100 AD-15136 3251 fCfAGCfTsT GAAccaTsT 3233- UgGuUcCcUgAgGaCcAgCTsT 1101 GCUGGucCUcaGG 1102 AD-15146 3251 GAAccaTsT 3242- UCUUCUUACUUCACCCGGCTT 1103 GCCGGGUGAAGUA 1104 AD-15260 3260 AGAAGATT 3243- CUUCUUACUUCACCCGGCUTT 1105 AGCCGGGUGAAGU 1106 AD-15371 3261 AAGAAGTT 3244- UUCUUACUUCACCCGGCUGTT 1107 CAGCCGGGUGAAG 1108 AD-15372 3262 UAAGAATT 3262- GGGCUCCUCAUUUUUACGGTT 1109 CCGUAAAAAUGAG 1110 AD-15172 3280 GAGCCCTT 3263- GGCUCCUCAUUUUUACGGGTT 1111 CCCGUAAAAAUGA 1112 AD-15295 3281 GGAGCCTT 3264- GCUCCUCAUUUUUACGGGUTT 1113 ACCCGUAAAAAUG 1114 AD-15373 3282 AGGAGCTT 3265- CUCCUCAUUUUUACGGGUATT 1115 UACCCGUAAAAAU 1116 AD-15163 3283 GAGGAGTT 3266- UCCUCAUUUUUACGGGUAATT 1117 UUACCCGUAAAAA 1118 AD-15165 3284 UGAGGATT 3267- CCUCAUUUUUACGGGUAACTT 1119 GUUACCCGUAAAA 1120 AD-15374 3285 AUGAGGTT 3268- CUCAUUUUUACGGGUAACATT 1121 UGUUACCCGUAAA 1122 AD-15296 3286 AAUGAGTT 3270- CAUUUUUACGGGUAACAGUTT 1123 ACUGUUACCCGUA 1124 AD-15261 3288 AAAAUGTT 3271- AUUUUUACGGGUAACAGUGTT 1125 CACUGUUACCCGU 1126 AD-15375 3289 AAAAAUTT 3274- UUUACGGGUAACAGUGAGGTT 1127 CCUCACUGUUACC 1128 AD-15262 3292 CGUAAATT 3308- CAGACCAGGAAGCUCGGUGTT 1129 CACCGAGCUUCCU 1130 AD-15376 3326 GGUCUGTT 3310- GACCAGGAAGCUCGGUGAGTT 1131 CUCACCGAGCUUC 1132 AD-15377 3328 CUGGUCTT 3312- CCAGGAAGCUCGGUGAGUGTT 1133 CACUCACCGAGCU 1134 AD-15409 3330 UCCUGGTT 3315- GGAAGCUCGGUGAGUGAUGTT 1135 CAUCACUCACCGA 1136 AD-15378 3333 GCUUCCTT 3324- GUGAGUGAUGGCAGAACGATT 1137 UCGUUCUGCCAUC 1138 AD-15410 3342 ACUCACTT 3326- GAGUGAUGGCAGAACGAUGTT 1139 CAUCGUUCUGCCA 1140 AD-15379 3344 UCACUCTT 3330- GAUGGCAGAACGAUGCCUGTT 1141 CAGGCAUCGUUCU 1142 AD-15187 3348 GCCAUCTT 3336- AGAACGAUGCCUGCAGGCATT 1143 UGCCUGCAGGCAU 1144 AD-15263 3354 CGUUCUTT 3339- ACGAUGCCUGCAGGCAUGGTT 1145 CCAUGCCUGCAGG 1146 AD-15264 3357 CAUCGUTT 3348- GCAGGCAUGGAACUUUUUCTT 1147 GAAAAAGUUCCAU 1148 AD-15297 3366 GCCUGCTT 3356- GGAACUUUUUCCGUUAUCATT 1149 UGAUAACGGAAAA 1150 AD-15208 3374 AGUUCCTT 3357- GAACUUUUUCCGUUAUCACTT 1151 GUGAUAACGGAAA 1152 AD-15209 3375 AAGUUCTT 3358- AACUUUUUCCGUUAUCACCTT 1153 GGUGAUAACGGAA 1154 AD-15193 3376 AAAGUUTT 3370- UAUCACCCAGGCCUGAUUCTT 1155 GAAUCAGGCCUGG 1156 AD-15380 3388 GUGAUATT 3378- AGGCCUGAUUCACUGGCCUTT 1157 AGGCCAGUGAAUC 1158 AD-15298 3396 AGGCCUTT 3383- UGAUUCACUGGCCUGGCGGTT 1159 CCGCCAGGCCAGU 1160 AD-15299 3401 GAAUCATT 3385- AUUCACUGGCCUGGCGGAGTT 1161 CUCCGCCAGGCCA 1162 AD-15265 3403 GUGAAUTT 3406- GCUUCUAAGGCAUGGUCGGTT 1163 CCGACCAUGCCUU 1164 AD-15381 3424 AGAAGCTT 3407- CUUCUAAGGCAUGGUCGGGTT 1165 CCCGACCAUGCCU 1166 AD-15210 3425 UAGAAGTT 3429- GAGGGCCAACAACUGUCCCTT 1167 GGGACAGUUGUUG 1168 AD-15270 3447 GCCCUCTT 3440- ACUGUCCCUCCUUGAGCACTsT 1169 GUGCUCAAGGAGG 1170 AD-9591 3458 GACAGUTsT 3440- AcuGucccuccuuGAGcAcTsT 1171 GUGCUcAAGGAGG 1172 AD-9717 3458 GAcAGUTsT 3441- CUGUCCCUCCUUGAGCACCTsT 1173 GGUGCUCAAGGAG 1174 AD-9622 3459 GGACAGTsT 3441- cuGucccuccuuGAGcAccTsT 1175 GGUGCUcAAGGAG 1176 AD-9748 3459 GGAcAGTsT 3480- ACAUUUAUCUUUUGGGUCUTsT 1177 AGACCCAAAAGAU 1178 AD-9587 3498 AAAUGUTsT 3480- AcAuuuAucuuuuGGGucuTsT 1179 AGACCcAAAAGAu 1180 AD-9713 3498 AAAUGUTsT 3480- AfcAfuUfuAfuCfuUfuUfgG 1181 P*aGfaCfcCfaA 1182 AD-14679 3498 fgUfcUfTsT faAfgAfuAfaAf uGfuTsT 3480- ACfAUfUfUfAUfCfUfUfUfU 1183 AGACfCfCfAAAA 1184 AD-14689 3498 fGGGUfCfUfTsT GAUfAAAUfGUfT sT 3480- AcAuUuAuCuUuUgGgUcUTsT 1185 P*aGfaCfcCfaA 1186 AD-14699 3498 faAfgAfuAfaAf uGfuTsT 3480- AcAuUuAuCuUuUgGgUcUTsT 1187 AGACfCfCfAAAA 1188 AD-14709 3498 GAUfAAAUfGUfT sT 3480- AfcAfuUfuAfuCfuUfuUfgG 1189 AGACCcaAAagAU 1190 AD-14719 3498 fgUfcUfTsT AAAuguTsT 3480- ACfAUfUfUfAUfCfUfUfUfU 1191 AGACCcaAAagAU 1192 AD-14729 3498 fGGGUfCfUfTsT AAAuguTsT 3480- AcAuUuAuCuUuUgGgUcUTsT 1193 AGACCcaAAagAU 1194 AD-14739 3498 AAAuguTsT 3480- GfcCfaUfcUfgCfuGfcCfgG 1195 P*gGfcUfcCfgG 1196 AD-15085 3498 faGfcCfTsT fcAfgCfaGfaUf gGfcTsT 3480- GCfCfAUfCfUfGCfUfGCfCf 1197 GGCfUfCfCfGGC 1198 AD-15095 3498 GGAGCfCfTsT fAGCfAGAUfGGC fTsT 3480- GcCaUcUgCuGcCgGaGcCTsT 1199 P*gGfcUfcCfgG 1200 AD-15105 3498 fcAfgCfaGfaUf gGfcTsT 3480- GcCaUcUgCuGcCgGaGcCTsT 1201 GGCfUfCfCfGGC 1202 AD-15115 3498 fAGCfAGAUfGGC fTsT 3480- GfcCfaUfcUfgCfuGfcCfgG 1203 GGCUCauGCagCA 1204 AD-15125 3498 faGfcCfTsT GAUggcTsT 3480- GCfCfAUfCfUfGCfUfGCfCf 1205 GGCUCauGCagCA 1206 AD-15135 3498 GGAGCfCfTsT GAUggcTsT 3480- GcCaUcUgCuGcCgGaGcCTsT 1207 GGCUCauGCagCA 1208 AD-15145 3498 GAUggcTsT 3481- CAUUUAUCUUUUGGGUCUGTsT 1209 CAGACCCAAAAGA 1210 AD-9578 3499 UAAAUGTsT 3481- cAuuuAucuuuuGGGucuGTsT 1211 cAGACCcAAAAGA 1212 AD-9704 3499 uAAAUGTsT 3485- UAUCUUUUGGGUCUGUCCUTsT 1213 AGGACAGACCCAA 1214 AD-9558 3503 AAGAUATsT 3485- uAucuuuuGGGucuGuccuTsT 1215 AGGAcAGACCcAA 1216 AD-9684 3503 AAGAuATsT 3504- CUCUGUUGCCUUUUUACAGTsT 1217 CUGUAAAAAGGCA 1218 AD-9634 3522 ACAGAGTsT 3504- cucuGuuGccuuuuuAcAGTsT 1219 CUGuAAAAAGGcA 1220 AD-9760 3522 AcAGAGTsT 3512- CCUUUUUACAGCCAACUUUTT 1221 AAAGUUGGCUGUA 1222 AD-15411 3530 AAAAGGTT 3521- AGCCAACUUUUCUAGACCUTT 1223 AGGUCUAGAAAAG 1224 AD-15266 3539 UUGGCUTT 3526- ACUUUUCUAGACCUGUUUUTT 1225 AAAACAGGUCUAG 1226 AD-15382 3544 AAAAGUTT 3530- UUCUAGACCUGUUUUGCUUTsT 1227 AAGCAAAACAGGU 1228 AD-9554 3548 CUAGAATsT 3530- uucuAGAccuGuuuuGcuuTsT 1229 AAGcAAAAcAGGU 1230 AD-9680 3548 CuAGAATsT 3530- UfuCfuAfgAfcCfuGfuUfuU 1231 P*aAfgCfaAfaA 1232 AD-14676 3548 fgCfuUfTsT fcAfgGfuCfuAf gAfaTsT 3530- UfUfCfUfAGACfCfUfGUfUf 1233 AAGCfAAAACfAG 1234 AD-14686 3548 UfUfGCfUfUfTsT GUfCfUfAGAATs T 3530- UuCuAgAcCuGuUuUgCuUTsT 1235 P*aAfgCfaAfaA 1236 AD-14696 3548 fcAfgGfuCfuAf gAfaTsT 3530- UuCuAgAcCuGuUuUgCuUTsT 1237 AAGCfAAAACfAG 1238 AD-14706 3548 GUfCfUfAGAATs T 3530- UfuCfuAfgAfcCfuGfuUfuU 1239 AAGcAaaACagGU 1240 AD-14716 3548 ffCfuUfTsT CUAgaaTsT 3530- UfUfCfUfAGACfCfUfGUfUf 1241 AAGcAaaACagGU 1242 AD-14726 3548 UfUfGCfUfUfTsT CUAgaaTsT 3530- UuCuAgAcCuGuUuUgCuUTsT 1243 AAGcAaaACagGU 1244 AD-14736 3548 CUAgaaTsT 3530- CfaUfaGfgCfcUfgGfaGfuU 1245 P*aAfuAfaAfcU 1246 AD-15082 3548 fuAfuUfTsT fcCfaGfgCfcUf aUfgTsT 3530- CfAUfAGGCfCfUfGGAGUfUf 1247 AAUfAAACfUfCf 1248 AD-15092 3548 UfAUfUfTsT CfAGGCfCfUfAU fGTsT 3530- CaUaGgCcUgGaGuUuAuUTsT 1249 P*aAfuAfaAfcU 1250 AD-15102 3548 fcCfaGfgCfcUf aUfgTsT 3530- CaUaGgCcUgGaGuUuAuUTsT 1251 AAUfAAACfUfCf 1252 AD-15112 3548 CfAGGCfCfUfAU fGTsT 3530- CfaUfaGfgCfcUfgGfaGfuU 1253 AAUAAacUCcaGG 1254 AD-15122 3548 fuAfuUfTsT CCUaugTsT 3530- CfAUfAGGCfCfUfGGAGUfUf 1255 AAUAAacUCcaGG 1256 AD-15132 3548 UfAUfUfTsT CCUaugTsT 3530- CaUaGgCcUgGaGuUuAuUTsT 1257 AAUAAacUCcaGG 1258 AD-15142 3548 CCUaugTsT 3531- UCUAGACCUGUUUUGCUUUTsT 1259 AAAGCAAAACAGG 1260 AD-9553 3549 UCUAGATsT 3531- ucuAGAccuGuuuuGcuuuTsT 1261 AAAGcAAAAcAGG 1262 AD-9679 3549 UCuAGATsT 3531- UfcUfaGfaCfcUfgUfuUfuG 1263 P*aAfaGfcAfaA 1264 AD-14675 3549 fcUfuUfTsT faCfaGfgUfcUf aGfaTsT 3531- UfCfUfAGACfCfUfGUfUfUf 1265 AAAGCfAAAACfA 1266 AD-14685 3549 UfGCfUfUfUfTsT GGUfCfUfAGATs T 3531- UcUaGaCcUgUuUuGcUuUTsT 1267 P*aAfaGfcAfaA 1268 AD-14695 3549 faCfaGfgUfcUf aGfaTsT 3531- UcUaGaCcUgUuUuGcUuUTsT 1269 AAAGCfAAAACfA 1270 AD-14705 3549 GGUfCfUfAGATs T 3531- UfcUfaGfaCfcUfgUfuUfuG 1271 AAAGCaaAAcaGG 1272 AD-14715 3549 fcUfuUfTsT UCUagaTsT 3531- UfCfUfAGACfCfUfGUfUfUf 1273 AAAGCaaAAcaGG 1274 AD-14725 3549 UfGCfUfUfUfTsT UCUagaTsT 3531- UcUaGaCcUgUuUuGcUuUTsT 1275 AAAGCaaAAcaGG 1276 AD-14735 3549 UCUagaTsT 3531- UfcAfuAfgGfcCfuGfgAfgU 1277 P*aUfaAfaCfuC 1278 AD-15081 3549 fuUfaUfTsT fcAfgGfcCfuAf uGfaTsT 3531- UfCfAUfAGGCfCfUfGGAGUf 1279 AUfAAACfUfCfC 1280 AD-15091 3549 UfUfAUfTsT fAGGCfCfUfAUf GATsT 3531- UcAuAgGcCuGgAgUuUaUTsT 1281 P*aUfaAfaCfuC 1282 AD-15101 3549 fcAfgGfcCfuAf uGfaTsT 3531- UcAuAgGcCuGgAgUuUaUTsT 1283 AUfAAACfUfCfC 1284 AD-15111 3549 fAGGCfCfUfAUf GATsT 3531- UfcAfuAfgGfcCfuGfgAfgU 1285 AUAAAcuCCagGC 1286 AD-15121 3549 fuUfaUfTsT CUAugaTsT 3531- UfCfAUfAGGCfCfUfGGAGUf 1287 AUAAAcuCCagGC 1288 AD-15131 3549 UfUfAUfTsT CUAugaTsT 3531- UcAuAgGcCuGgAgUuUaUTsT 1289 AUAAAcuCCagGC 1290 AD-15141 3549 CUAugaTsT 3557- UGAAGAUAUUUAUUCUGGGTsT 1291 CCCAGAAUAAAUA 1292 AD-9626 3575 UCUUCATsT 3557- uGAAGAuAuuuAuucuGGGTsT 1293 CCcAGAAuAAAuA 1294 AD-9752 3575 UCUUcATsT 3570- UCUGGGUUUUGUAGCAUUUTsT 1295 AAAUGCUACAAAA 1296 AD-9629 3588 CCCAGATsT 3570- ucuGGGuuuuGuAGcAuuuTsT 1297 AAAUGCuAcAAAA 1298 AD-9755 3588 CCcAGATsT 3613- AUAAAAACAAACAAACGUUTT 1299 AACGUUUGUUUGU 1300 AD-15412 3631 UUUUAUTT 3617- AAACAAACAAACGUUGUCCTT 1301 GGACAACGUUUGU 1302 AD-15211 3635 UUGUUUTT 3618- AACAAACAAACGUUGUCCUTT 1303 AGGACAACGUUUG 1304 AD-15300 3636 UUUGUUTT *Target: target in human PCSK9 gene, access. # NM_174936 U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide.

TABLE 5 Sequences of modified dsRNA targeted to PCSK9 SEQ Sense strand sequence (5′- SEQ ID Antisense-strand ID Duplex # 3′)¹ NO: sequence (5′-3′)¹ NO: AD-10792 GccuGGAGuuuAuucGGAATsT 1305 UUCCGAAuAAACUCcAGGCT 1306 sT AD-10793 GccuGGAGuuuAuucGGAATsT 1307 uUcCGAAuAAACUccAGGCT 1308 sT AD-10796 GccuGGAGuuuAuucGGAATsT 1309 UUCCGAAUAAACUCCAGGCT 1310 sT AD-12038 GccuGGAGuuuAuucGGAATsT 1311 uUCCGAAUAAACUCCAGGCT 1312 sT AD-12039 GccuGGAGuuuAuucGGAATsT 1313 UuCCGAAUAAACUCCAGGCT 1314 sT AD-12040 GccuGGAGuuuAuucGGAATsT 1315 UUcCGAAUAAACUCCAGGCT 1316 sT AD-12041 GccuGGAGuuuAuucGGAATsT 1317 UUCcGAAUAAACUCCAGGCT 1318 sT AD-12042 GCCUGGAGUUUAUUCGGAATsT 1319 uUCCGAAUAAACUCCAGGCT 1320 sT AD-12043 GCCUGGAGUUUAUUCGGAATsT 1321 UuCCGAAUAAACUCCAGGCT 1322 sT AD-12044 GCCUGGAGUUUAUUCGGAATsT 1323 UUcCGAAUAAACUCCAGGCT 1324 sT AD-12045 GCCUGGAGUUUAUUCGGAATsT 1325 UUCcGAAUAAACUCCAGGCT 1326 sT AD-12046 GccuGGAGuuuAuucGGAA 1327 UUCCGAAUAAACUCCAGGCs 1328 csu AD-12047 GccuGGAGuuuAuucGGAAA 1329 UUUCCGAAUAAACUCCAGGC 1330 scsu AD-12048 GccuGGAGuuuAuucGGAAAA 1331 UUUUCCGAAUAAACUCCAGG 1332 Cscsu AD-12049 GccuGGAGuuuAuucGGAAAAG 1333 CUUUUCCGAAUAAACUCCAG 1334 GCscsu AD-12050 GccuGGAGuuuAuucGGAATTab 1335 UUCCGAAUAAACUCCAGGCT 1336 Tab AD-12051 GccuGGAGuuuAuucGGAAATTab 1337 UUUCCGAAuAAACUCCAGGC 1338 TTab AD-12052 GccuGGAGuuuAuucGGAAAATTab 1339 UUUUCCGAAUAAACUCCAGG 1340 CTTab AD-12053 GccuGGAGuuuAuucGGAAAAGTTab 1341 CUUUUCCGAAUAAACUCCAG 1342 GCTTab AD-12054 GCCUGGAGUUUAUUCGGAATsT 1343 UUCCGAAUAAACUCCAGGCs 1344 csu AD-12055 GccuGGAGuuuAuucGGAATsT 1345 UUCCGAAUAAACUCCAGGCs 1346 csu AD-12056 GcCuGgAgUuUaUuCgGaA 1347 UUCCGAAUAAACUCCAGGCT 1348 Tab AD-12057 GcCuGgAgUuUaUuCgGaA 1349 UUCCGAAUAAACUCCAGGCT 1350 sT AD-12058 GcCuGgAgUuUaUuCgGaA 1351 UUCCGAAuAAACUCcAGGCT 1352 sT AD-12059 GcCuGgAgUuUaUuCgGaA 1353 uUcCGAAuAAACUccAGGCT 1354 sT AD-12060 GcCuGgAgUuUaUuCgGaA 1355 UUCCGaaUAaaCUCCAggc 1356 AD-12061 GcCuGgnAgUuUaUuCgGaATsT 1357 UU CCGaaUAaaCUCCAggcT 1358 sT AD-12062 GcCuGgAgUuUaUuCgGaATTab 1359 UUCCGaaUAaaCUCCAggcT 1360 Tab AD-12063 GcCuGgAgUuUaUuCgGaA 1361 UUCCGaaUAaaCUCCAggcs 1362 csu AD-12064 GcCuGgnAgUuUaUuCgGaATsT 1363 UU CCGAAuAAACUCcAGGCT 1364 sT AD-12065 GcCuGgAgUuUaUuCgGaATTab 1365 UUCCGAAuAAACUCcAGGCT 1366 Tab AD-12066 GcCuGgAgUuUaUuCgGaA 1367 UUCCGAAuAAACUCcAGGCs 1368 csu AD-12067 GcCuGgnAgUuUaUuCgGaATsT 1369 UUCCGAAUAAACUCCAGGCT 1370 sT AD-12068 GcCuGgAgUuUaUuCgGaATTab 1371 UUCCGAAUAAACUCCAGGCT 1372 Tab AD-12069 GcCuGgAgUuUaUuCgGaA 1373 UUCCGAAUAAACUCCAGGCs 1374 csu AD-12338 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1375 P*uUfcCfgAfaUfaAfaCf 1376 f uCfcAfgGfc AD-12339 GcCuGgAgUuUaUuCgGaA 1377 P*uUfcCfgAfaUfaAfaCf 1378 uCfcAfgGfc AD-12340 GccuGGAGuuuAuucGGAA 1379 P*uUfcCfgAfaUfaAfaCf 1380 uCfcAfgGfc AD-12341 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1381 P*uUfcCfgAfaUfaAfaCf 1382 fTsT uCfcAfgGfcTsT AD-12342 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1383 UUCCGAAuAAACUCcAGGCT 1384 fTsT sT AD-12343 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1385 uUcCGAAuAAACUccAGGCT 1386 fTsT sT AD-12344 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1387 UsTUCCGAAUAAACUCCAGGCT 1388 fTsT sT AD-12345 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1389 UUCCGAAUAAACUCCAGGCs 1390 fTsT csu AD-12346 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1391 UUCCGaaUAaaCUCCAggcs 1392 fTsT csu AD-12347 GCCUGGAGUUUAUUCGGAATsT 1393 P*uUfcCfgAfaUfaAfaCf 1394 uCfcAfgGfcTsT AD-12348 GccuGGAGuuuAuucGGAATsT 1395 P*uUfcCfgAfaUfaAfaCf 1396 uCfcAfgGfcTsT AD-12349 GcCuGgnAgUuUaUuCgGaATsT 1397 P*uUfcCfgAfaUfaAfaCf 1398 uCfcAfgGfcTsT AD-12350 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1399 P*uUfcCfgAfaUfaAfaCf 1400 fTTab uCfcAfgGfcTTab AD-12351 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1401 P*uUfcCfgAfaUfaAfaCf 1402 f uCfcAfgGfcsCfsu AD-12352 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1403 UUCCGaaUAaaCUCCAggcs 1404 f csu AD-12354 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1405 UUCCGAAUAAACUCCAGGCs 1406 f csu AD-12355 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1407 UUCCGAAuAAACUCcAGGCT 1408 f sT AD-12356 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1409 uUcCGAAuAAACUccAGGCT 1410 f sT AD-12357 GmocCmouGmogAm02gUmouUmoaUmo 1411 UUCCGaaUAaaCUCCAggc 1412 uCmogGmoaA AD-12358 GmocCmouGmogAm02gUmouUmoaUmo 1413 P*uUfcCfgAfaUfaAfaCf 1414 uCmogGmoaA uCfcAfgGfc AD-12359 GmocCmouGmogAm02gUmouUmoaUmo 1415 P*uUfcCfgAfaUfaAfaCf 1416 uCmogGmoaA uCfcAfgGfcsCfsu AD-12360 GmocCmouGmogAm02gUmouUmoaUmo 1417 UUCCGAAUAAACUCCAGGCs 1418 uCmogGmoaA csu AD-12361 GmocCmouGmogAm02gUmouUmoaUmo 1419 UUCCGAAuAAACUCcAGGCT 1420 uCmogGmoaA sT AD-12362 GmocCmouGmogAm02gUmouUmoaUmo 1421 uUcCGAAuAAACUccAGGCT 1422 uCmogGmoaA sT AD-12363 GmocCmouGmogAm02gUmouUmoaUmo 1423 UUCCGaaUAaaCUCCAggcs 1424 uCmogGmoaA csu AD-12364 GmocCmouGmogAmogUmouUmoaUmou 1425 UUCCGaaUAaaCUCCAggcT 1426 CmogGmoaATsT sT AD-12365 GmocCmouGmogAmogUmouUmoaUmou 1427 UUCCGAAuAAACUCcAGGCT 1428 CmogGmoaATsT sT AD-12366 GmocCmouGmogAmogUmouUmoaUmou 1429 UUCCGAAUAAACUCCAGGCT 1430 CmogGmoaATsT sT AD-12367 GmocmocmouGGAGmoumoumouAmoum 1431 UUCCGaaUAaaCUCCAggcT 1432 oumocGGAATsT sT AD-12368 GmocmocmouGGAGmoumoumouAmoum 1433 UUCCGAAuAAACUCcAGGCT 1434 oumocGGAATsT sT AD-12369 GmocmocmouGGAGmoumoumouAmoum 1435 UUCCGAAUAAACUCCAGGCT 1436 oumocGGAATsT sT AD-12370 GmocmocmouGGAGmoumoumouAmoum 1437 P*UfUfCfCfGAAUfAAACf 1438 umocGGAATsT UfCfCfAGGCfTsT AD-12371 GmocmocmouGGAGmoumoumouAmoum 1439 P*UfUfCfCfGAAUfAAACf 1440 umocGGAATsT UfCfCfAGGCfsCfsUf AD-12372 GmocmocmouGGAGmoumoumouAmoum 1441 P*uUfcCfgAfaUfaAfaCf 1442 oumocGGAATsT uCfcAfgGfcsCfsu AD-12373 GmocmocmouGGAGmoumoumouAmoum 1443 UUCCGAAUAAACUCCAGGCT 1444 oumocGGAATsT sT AD-12374 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1445 UfUfCfCfGAAUfAAACfUf 1446 TsT CfCfAGGCfTsT AD-12375 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1447 UUCCGAAUAAACUCCAGGCT 1448 TsT sT AD-12377 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1449 uUcCGAAuAAACUccAGGCT 1450 TsT sT AD-12378 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1451 UUCCGaaUAaaCUCCAggcs 1452 TsT csu AD-12379 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1453 UUCCGAAUAAACUCCAGGCs 1454 TsT csu AD-12380 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1455 P*uUfcCfgAfaUfaAfaCf 1456 TsT uCfcAfgGfcsCfsu AD-12381 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1457 P*uUfcCfgAfaUfaAfaCf 1458 TsT uCfcAfgGfcTsT AD-12382 GCfCfUfGGAGUfUfUfAUfUfCfGGAA 1459 P*UfUfCfCfGAAUfAAACf 1460 TsT UfCfCfAGGCfTsT AD-12383 GCCUGGAGUUUAUUCGGAATsT 1461 P*UfUfCfCfGAAUfAAACf 1462 UfCfCfAGGCfTsT AD-12384 GccuGGAGuuuAuucGGAATsT 1463 P*UfUfCfCfGAAUfAAACf 1464 UfCfCfAGGCfTsT AD-12385 GcCuGgnAgUuUaUuCgGaATsT 1465 P*UfUfCfCfGAAUfAAACf UfCfCfAGGCfTsT AD-12386 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1467 P*UfUfCfCfGAAUfAAACf 1468 f UfCfCfAGGCfTsT AD-12387 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1469 UfUfCfCfGAAUfAAACfUf 1470 A CfCfAGGCfsCfsUf AD-12388 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1471 P*uUfcCfgAfaUfaAfaCf 1472 A uCfcAfgGfc AD-12389 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1473 P*uUfcCfgAfaUfaAfaCf 1474 A uCfcAfgGfcsCfsu AD-12390 GCfCfUfGGAGGUfUfUfAUfUfCfGGAA 1475 UUCCGAAUAAACUCCAGGCs 1476 A csu AD-12391 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1477 UUCCGaaUAaaCUCCAggc 1478 A AD-12392 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1479 UUCCGAAUAAACUCCAGGCT 1480 A sT AD-12393 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1481 UUCCGAAuAAACUCcAGGCT 1482 A sT AD-12394 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1483 uUcCGAAuAAACUccAGGCT 1484 A sT AD-12395 GmocCmouGmogAmogUmouUmoaUmou 1485 P*UfUfCfCfGAAUfAAACf 1486 CmogGmoaATsT UfCfCfAGGCfsCfsUf AD-12396 GmocCmouGmogAm02gUmouUmoaUmo 1487 P*UfUfCfCfGAAUfAAACf 1488 uCmogGmoaA UfCfCfAGGCfsCfsUf AD-12397 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1489 P*UfUfCfCfGAAUfAAACf 1490 f UfCfCfAGGCfsCfsUf AD-12398 GfcCfuGfgAfgUfuUfaUfuCfgGfaA 1491 P*UfUfCfCfGAAUfAAACf 1492 fTsT UfCfCfAGGCfsCfsUf AD-12399 GcCuGgnAgUuUaUuCgGaATsT 1493 P*UfUfCfCfGAAUfAAACf 1494 UfCfCfAGGCfsCfsUf AD-12400 GCCUGGAGUUUAUUCGGAATsT 1495 P*UfUfCfCfGAAUfAAACf 1496 UfCfCfAGGCfsCfsUf AD-12401 GccuGGAGuuuAuucGGAATsT 1497 P*UfUfCfCfGAAUfAAACf 1498 UfCfCfAGGCfsCfsUf AD-12402 GccuGGAGuuuAuucGGAA 1499 P*UfUfCfCfGAAUfAAACf 1500 UfCfCfAGGCfsCfsUf AD-12403 GCfCfUfGGAGGUfUfUfAUfUfCfGGA 1501 P*UfUfCfCfGAAUfAAACf 1502 A UfCfCfAGGCfsCfsUf AD-9314 GCCUGGAGUUUAUUCGGAATsT 1503 UUCCGAAUAAACUCCAGGCT 1504 sT AD-10794 ucAuAGGccuGGAGuuuAudTsdT 1525 AuAAACUCcAGGCCuAUGAd 1526 TsdT AD-10795 ucAuAGGccuGGAGuuuAudTsdT 1527 AuAAACUccAGGcCuAuGAd 1528 TsdT AD-10797 ucAuAGGccuGGAGuuuAudTsdT 1529 AUAAACUCCAGGCCUAUGAd 1530 TsdT U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide.

TABLE 6 dsRNA tarteted to PCSK9: mismatches and modifications Du- SEQ plex ID # Strand NO: Sequence (5′ to 3′) AD- S 1531 uucuAGAccuGuuuuGcuudTsdT 9680 AS 1532 AAGcAAAAcAGGUCuAGAAdTsdT AD- S 1535 uucuAGAcCuGuuuuGcuuTsT 3267 AS 1536 AAGcAAAAcAGGUCuAGAATsT AD- S 1537 uucuAGAccUGuuuuGcuuTsT 3268 AS 1538 AAGcAAAAcAGGUCuAGAATsT AD- S 1539 uucuAGAcCUGuuuuGcuuTsT 3269 AS 1540 AAGcAAAAcAGGUCuAGAATsT AD- S 1541 uucuAGAcYluGuuuuGcuuTsT 3270 AS 1542 AAGcAAAAcAGGUCuAGAATsT AD- S 1543 uucuAGAcYlUGuuuuGcuuTsT 3271 AS 1544 AAGcAAAAcAGGUCuAGAATsT AD- S 1545 uucuAGAccYlGuuuuGcuuTsT 3272 AS 1546 AAGcAAAAcAGGUCuAGAATsT AD- S 1547 uucuAGAcCYlGuuuuGcuuTsT 3273 AS 1548 AAGcAAAAcAGGUCuAGAATsT AD- S 1549 uucuAGAccuYluuuuGcuuTsT 3274 AS 1550 AAGcAAAAcAGGUCuAGAATsT AD- S 1551 uucuAGAcCUYluuuuGcuuTsT 3275 AS 1552 AAGcAAAAcAGGUCuAGAATsT AD- S 1553 UfuCfuAfgAfcCfuGfuUfuUfgCfuUfTsT 14676 AS 1554 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1555 UfuCfuAfgAfcCuGfuUfuUfgCfuUfTsT 3276 AS 1556 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1557 UfuCfuAfgAfcCfUGfuUfuUfgCfuUfTsT 3277 AS 1558 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1559 UfuCfuAfgAfcCUGfuUfuUfgCfuUfTsT 3278 AS 1560 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1561 UfuCfuAfgAfcYluGfuUfuUfgCfuUfTsT 3279 AS 1562 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1563 UfuCfuAfgAfcYlUGfuUfuUfgCfuUfTsT 3280 AS 1564 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1565 UfuCfuAfgAfcCfYlGfuUfuUfgCfuUfTsT 3281 AS 1566 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1567 UfuCfuAfgAfcCYlGfuUfuUfgCfuUfTsT 3282 AS 1568 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1569 UfuCfuAfgAfcCfuYluUfuUfgCfuUfTsT 3283 AS 1570 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 1571 UfuCfuAfgAfcCUYluUfuUfgCfuUfTsT 3284 AS 1572 P*aAfgCfaAfaAfcAfgGfuCfuAfgAfaTsT AD- S 459 GccuGGAGuuuAuucGGAATsT 10792 AS 460 UUCCGAAuAAACUCcAGGCTsT AD- S 1573 GccuGGAGuYluAuucGGAATsT 3254 AS 1574 UUCCGAAuAAACUCcAGGCTsT AD- S 1575 GccuGGAGUYluAuucGGAATsT 3255 AS 1576 UUCCGAAuAAACUCcAGGCTsT Strand: S/Sense; AS/Antisense; U, C, A, G: corresponding ribonucleotide; T: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Yl corresponds to DFT difluorotoluyl ribo(or deoxyribo)nucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

TABLE 7 Sequences of unmodified siRNA flanking AD-9680 SEQ *Tar- ID Duplex # Strand Sequence (5′ to 3′) get NO: AD-22169- sense CAGCCAACUUUUCUAGACCdTsdT 3520 1577 b1 antis GGUCUAGAAAAGUUGGCUGdTsdT 3520 1578 AD-22170- sense AGCCAACUUUUCUAGACCUdTsdT 3521 1579 b1 antis AGGUCUAGAAAAGUUGGCUdTsdT 3521 1580 AD-22171- sense GCCAACUUUUCUAGACCUGdTsdT 3522 1581 b1 antis CAGGUCUAGAAAAGUUGGCdTsdT 3522 1582 AD-22172- sense CCAACUUUUCUAGACCUGUdTsdT 3523 1583 b1 antis ACAGGUCUAGAAAAGUUGGdTsdT 3523 1584 AD-22173- sense CAACUUUUCUAGACCUGUUdTsdT 3524 1585 b1 antis AACAGGUCUAGAAAAGUUGdTsdT 3524 1586 AD-22174- sense AACUUUUCUAGACCUGUUUdTsdT 3525 1587 b1 antis AAACAGGUCUAGAAAAGUUdTsdT 3525 1588 AD-22175- sense ACUUUUCUAGACCUGUUUUdTsdT 3526 1589 b1 antis AAAACAGGUCUAGAAAAGUdTsdT 3526 1590 AD-22176- sense CUUUUCUAGACCUGUUUUGdTsdT 3527 1591 b1 antis CAAAACAGGUCUAGAAAAGdTsdT 3527 1592 AD-22177- sense UUUUCUAGACCUGUUUUGCdTsdT 3528 1593 b1 antis GCAAAACAGGUCUAGAAAAdTsdT 3528 1594 AD-22178- sense UUUCUAGACCUGUUUUGCUdTsdT 3529 1595 b1 antis AGCAAAACAGGUCUAGAAAdTsdT 3529 1596 AD-22179- sense UCUAGACCUGUUUUGCUUUdTsdT 3531 1597 b1 antis AAAGCAAAACAGGUCUAGAdTsdT 3531 1598 AD-22180- sense CUAGACCUGUUUUGCUUUUdTsdT 3532 1599 b1 antis AAAAGCAAAACAGGUCUAGdTsdT 3532 1600 AD-22181- sense UAGACCUGUUUUGCUUUUGdTsdT 3533 1601 b1 antis CAAAAGCAAAACAGGUCUAdTsdT 3533 1602 AD-22182- sense AGACCUGUUUUGCUUUUGUdTsdT 3534 1603 b1 antis ACAAAAGCAAAACAGGUCUdTsdT 3534 1604 AD-22183- sense GACCUGUUUUGCUUUUGUAdTsdT 3535 1605 b1 antis UACAAAAGCAAAACAGGUCdTsdT 3535 1606 AD-22184- sense ACCUGUUUUGCUUUUGUAAdTsdT 3536 1607 b1 antis UUACAAAAGCAAAACAGGUdTsdT 3536 1608 AD-22185- sense CCUGUUUUGCUUUUGUAACdTsdT 3537 1609 b1 antis GUUACAAAAGCAAAACAGGdTsdT 3537 1610 AD-22186- sense CUGUUUUGCUUUUGUAACUdTsdT 3538 1611 b1 antis AGUUACAAAAGCAAAACAGdTsdT 3538 1612 AD-22187- sense UGUUUUGCUUUUGUAACUUdTsdT 3539 1613 b1 antis AAGUUACAAAAGCAAAACAdTsdT 3539 1614 AD-22188- sense GUUUUGCUUUUGUAACUUGdTsdT 3540 1615 b1 antis CAAGUUACAAAAGCAAAACdTsdT 3540 1616 AD-22189- sense UUUUGCUUUUGUAACUUGAdTsdT 3541 1617 b1 antis UCAAGUUACAAAAGCAAAAdTsdT 3541 1618 AD-22190- sense UUUGCUUUUGUAACUUGAAdTsdT 3542 1619 b1 antis UUCAAGUUACAAAAGCAAAdTsdT 3542 1620 AD-22191- sense UUGCUUUUGUAACUUGAAGdTsdT 3543 1621 b1 antis CUUCAAGUUACAAAAGCAAdTsdT 3543 1622 AD-22192- sense UGCUUUUGUAACUUGAAGAdTsdT 3544 1623 b1 antis UCUUCAAGUUACAAAAGCAdTsdT 3544 1624 AD-22193- sense GCUUUUGUAACUUGAAGAUdTsdT 3545 1625 b1 antis AUCUUCAAGUUACAAAAGCdTsdT 3545 1626 AD-22194- sense CUUUUGUAACUUGAAGAUAdTsdT 3546 1627 b1 antis UAUCUUCAAGUUACAAAAGdTsdT 3546 1628 AD-22195- sense UUUUGUAACUUGAAGAUAUdTsdT 3547 1629 b1 antis AUAUCUUCAAGUUACAAAAdTsdT 3547 1630 AD-22196- sense UUUGUAACUUGAAGAUAUUdTsdT 3548 1631 b1 antis AAUAUCUUCAAGUUACAAAdTsdT 3548 1632 AD-22197- sense UUGUAACUUGAAGAUAUUUdTsdT 3549 1633 b1 antis AAAUAUCUUCAAGUUACAAdTsdT 3549 1634 AD-22198- sense UGUAACUUGAAGAUAUUUAdTsdT 3550 1635 b1 antis UAAAUAUCUUCAAGUUACAdTsdT 3550 1636 AD-22199- sense GUAACUUGAAGAUAUUUAUdTsdT 3551 1637 b1 antis AUAAAUAUCUUCAAGUUACdTsdT 3551 1638 AD-22200- sense UAACUUGAAGAUAUUUAUUdTsdT 3552 1639 b1 antis AAUAAAUAUCUUCAAGUUAdTsdT 3552 1640 AD-22201- sense AACUUGAAGAUAUUUAUUCdTsdT 3553 1641 b1 antis GAAUAAAUAUCUUCAAGUUdTsdT 3553 1642 AD-22202- sense ACUUGAAGAUAUUUAUUCUdTsdT 3554 1643 b1 antis AGAAUAAAUAUCUUCAAGUdTsdT 3554 1644 AD-22203- sense CUUGAAGAUAUUUAUUCUGdTsdT 3555 1645 b1 antis CAGAAUAAAUAUCUUCAAGdTsdT 3555 1646 AD-22204- sense UUGAAGAUAUUUAUUCUGGdTsdT 3556 1647 b1 antis CCAGAAUAAAUAUCUUCAAdTsdT 3556 1648 AD-22205- sense UGAAGAUAUUUAUUCUGGGdTsdT 3557 1649 b1 antis CCCAGAAUAAAUAUCUUCAdTsdT 3557 1650 AD-22206- sense GAAGAUAUUUAUUCUGGGUdTsdT 3558 1651 b1 antis ACCCAGAAUAAAUAUCUUCdTsdT 3558 1652 *Target: target in human PCSK9 gene, access. # NM_174936 U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups.

TABLE 8 Sequences of modified siRNA flankint AD-9680 SEQ *Tar- ID Duplex # Strand Sequence (5′ to 3′) get NO: AD-22098- sense cAGccAAcuuuucuAGAccdTsdT 3520 1653 b1 antis GGUCuAGAAAAGUUGGCUGdTsdT 3520 1654 AD-22099- sense AGccAAcuuuucuAGAccudTsdT 3521 1655 b1 antis AGGUCuAGAAAAGUUGGCUdTsdT 3521 1656 AD-22100- sense GccAAcuuuucuAGAccuGdTsdT 3522 1657 b1 antis cAGGUCuAGAAAAGUUGGCdTsdT 3522 1658 AD-22101- sense ccAAcuuuucuAGAccuGudTsdT 3523 1659 b1 antis AcAGGUCuAGAAAAGUUGGdTsdT 3523 1660 AD-22102- sense cAAcuuuucuAGAccuGuudTsdT 3524 1661 b1 antis AAcAGGUCuAGAAAAGUUGdTsdT 3524 1662 AD-22103- sense AAcuuuucuAGAccuGuuudTsdT 3525 1663 b1 antis AAAcAGGUCuAGAAAAGUUdTsdT 3525 1664 AD-22104- sense AcuuuucuAGAccuGuuuudTsdT 3526 1665 b1 antis AAAAcAGGUCuAGAAAAGUdTsdT 3526 1666 AD-22105- sense cuuuucuAGAccuGuuuuGdTsdT 3527 1667 b1 antis cAAAAcAGGUCuAGAAAAGdTsdT 3527 1668 AD-22106- sense uuuucuAGAccuGuuuuGcdTsdT 3528 1669 b1 antis GcAAAAcAGGUCuAGAAAAdTsdT 3528 1670 AD-22107- sense uuucuAGAccuGuuuuGcudTsdT 3529 1671 b1 antis AGcAAAAcAGGUCuAGAAAdTsdT 3529 1672 AD-22108- sense ucuAGAccuGuuuuGcuuudTsdT 3531 1673 b1 antis AAAGcAAAAcAGGUCuAGAdTsdT 3531 1674 AD-22109- sense cuAGAccuGuuuuGcuuuudTsdT 3532 1675 b1 antis AAAAGcAAAAcAGGUCuAGdTsdT 3532 1676 AD-22110- sense uAGAccuGuuuuGcuuuuGdTsdT 3533 1677 b1 antis cAAAAGcAAAAcAGGUCuAdTsdT 3533 1678 AD-22111- sense AGAccuGuuuuGcuuuuGudTsdT 3534 1679 b1 antis AcAAAAGcAAAAcAGGUCUdTsdT 3534 1680 AD-22112- sense GAccuGuuuuGcuuuuGuAdTsdT 3535 1681 b1 antis uAcAAAAGcAAAAcAGGUCdTsdT 3535 1682 AD-22113- sense AccuGuuuuGcuuuuGuAAdTsdT 3536 1683 b1 antis UuAcAAAAGcAAAAcAGGUdTsdT 3536 1684 AD-22114- sense ccuGuuuuGcuuuuGuAAcdTsdT 3537 1685 b1 antis GUuAcAAAAGcAAAAcAGGdTsdT 3537 1686 AD-22115- sense cuGuuuuGcuuuuGuAAcudTsdT 3538 1687 b1 antis AGUuAcAAAAGcAAAAcAGdTsdT 3538 1688 sense uGuuuuGcuuuuGuAAcuudTsdT 3539 1689 antis AAGUuAcAAAAGcAAAAcAdTsdT 3539 1690 AD-22116- sense GuuuuGcuuuuGuAAcuuGdTsdT 3540 1691 b1 antis cAAGUuAcAAAAGcAAAACdTsdT 3540 1692 AD-22117- sense uuuuGcuuuuGuAAcuuGAdTsdT 3541 1693 b1 antis UcAAGUuAcAAAAGcAAAAdTsdT 3541 1694 AD-22118- sense uuuGcuuuuGuAAcuuGAAdTsdT 3542 1695 b1 antis UUcAAGUuAcAAAAGcAAAdTsdT 3542 1696 AD-22119- sense uuGcuuuuGuAAcuuGAAGdTsdT 3543 1697 b1 antis CUUcAAGUuAcAAAAGcAAdTsdT 3543 1698 AD-22120- sense uGcuuuuGuAAcuuGAAGAdTsdT 3544 1699 b1 antis UCUUcAAGUuAcAAAAGcAdTsdT 3544 1700 AD-22121- sense GcuuuuGuAAcuuGAAGAudTsdT 3545 1701 b1 antis AUCUUcAAGUuAcAAAAGCdTsdT 3545 1702 AD-22122- sense cuuuuGuAAcuuGAAGAuAdTsdT 3546 1703 b1 antis uAUCUUcAAGUuAcAAAAGdTsdT 3546 1704 AD-22123- sense uuuuGuAAcuuGAAGAuAudTsdT 3547 1705 b1 antis AuAUCUUcAAGUuAcAAAAdTsdT 3547 1706 AD-22124- sense uuuGuAAcuuGAAGAuAuudTsdT 3548 1707 b1 antis AAuAUCUUcAAGUuAcAAAdTsdT 3548 1708 AD-22125- sense uuGuAAcuuGAAGAuAuuudTsdT 3549 1709 b1 antis AAAuAUCUUcAAGUuAcAAdTsdT 3549 1710 AD-22126- sense uGuAAcuuGAAGAuAuuuAdTsdT 3550 1711 b1 antis uAAAuAUCUUcAAGUuAcAdTsdT 3550 1712 AD-22127- sense GuAAcuuGAAGAuAuuuAudTsdT 3551 1713 b1 antis AuAAAuAUCUUcAAGUuACdTsdT 3551 1714 AD-22128- sense uAAcuuGAAGAuAuuuAuudTsdT 3552 1715 b1 antis AAuAAAuAUCUUcAAGUuAdTsdT 3552 1716 AD-22129- sense AAcuuGAAGAuAuuuAuucdTsdT 3553 1717 b1 antis GAAuAAAuAUCUUcAAGUUdTsdT 3553 1718 AD-22130- sense AcuuGAAGAuAuuuAuucudTsdT 3554 1719 b1 antis AGAAuAAAuAUCUUcAAGUdTsdT 3554 1720 AD-22131- sense cuuGAAGAuAuuuAuucuGdTsdT 3555 1721 b1 antis cAGAAuAAAuAUCUUcAAGdTsdT 3555 1722 AD-22132- sense uuGAAGAuAuuuAuucuGGdTsdT 3556 1723 b1 antis CcAGAAuAAAuAUCUUcAAdTsdT 3556 1724 AD-22133- sense uGAAGAuAuuuAuucuGGGdTsdT 3557 1725 b1 antis CCcAGAAuAAAuAUCUUcAdTsdT 3557 1726 AD-22134- sense GAAGAuAuuuAuucuGGGudTsdT 3558 1727 b1 antis ACCcAGAAuAAAuAUCUUCdTsdT 3558 1728 *Target: 5′ nuticoetide of target sequence in human PCSK9 gene, access. # NM_174936 U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; Uf, Cf, Af, Gf: corresponding 2′-deoxy-2′-fluoro ribonucleotide; Yl corresponds to DFT difluorotoluyl ribo(or deoxyribo)nucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups; unless denoted by prefix “P*”, oligonucleotides are devoid of a 5′-phosphate group on the 5′-most nucleotide; all oligonucleotides bear 3′-OH on the 3′-most nucleotide

TABLE 9 Sequences of XBP-1 dsRNAs SEQ ID SEQ ID Target* NO sense (5′-3′) NO antisense (5′-3′) NM_0010042101128- 1729 CCCAGCUGAUUAGUGUCUA 1753 UAGACACUAAUCAGCUGGG 1146 NM_0010042101129- 1730 CCAGCUGAUUAGUGUCUAA 1754 UUAGACACUAAUCAGCUGG 1147 NM_001004210677- 1731 CUCCCAGAGGUCUACCCAG 1755 CUGGGUAGACCUCUGGGAG 695 NM_001004210893- 1732 GAUCACCCUGAAUUCAUUG 1756 CAAUGAAUUCAGGGUGAUC 911 NM_001004210895- 1733 UCACCCUGAAUUCAUUGUC 1757 GACAAUGAAUUCAGGGUGA 913 NM_0010042101127- 1734 CCCCAGCUGAUUAGUGUCU 1758 AGACACUAAUCAGCUGGGG 1145 NM_001004210894- 1735 AUCACCCUGAAUUCAUUGU 1759 ACAAUGAAUUCAGGGUGAU 912 NM_0010042101760- 1736 CAUUUAUUUAAAACUACCC 1760 GGGUAGUUUUAAAUAAAUG 1778 NM_001004210215- 1737 ACUGAAAAACAGAGUAGCA 1761 UGCUACUCUGUUUUUCAGU 233 NM_0010042101759- 1738 CCAUUUAUUUAAAACUACC 1762 GGUAGUUUUAAAUAAAUGG 1777 NM_001004210367- 1739 UUGAGAACCAGGAGUUAAG 1763 CUUAACUCCUGGUUCUCAA 385 NM_001004210896- 1740 CACCCUGAAUUCAUUGUCU 1764 AGACAAUGAAUUCAGGGUG 914 NM_001004210214- 1741 AACUGAAAAACAGAGUAGC 1765 GCUACUCUGUUUUUCAGUU 232 NM_001004210216- 1742 CUGAAAAACAGAGUAGCAG 1766 CUGCUACUCUGUUUUUCAG 234 XM_001103095387- 1743 AGAAAAUCAGCUUUUACGA 1767 UCGUAAAAGCUGAUUUUCU 405 XM_0011030951151- 1744 UCCCCAGCUGAUUAGUGUC 1768 GACACUAAUCAGCUGGGGA 1169 XM_0011030951466- 1745 UACUUAUUAUGUAAGGGUC 1769 GACCCUUACAUAAUAAGUA 1484 XM_0011030951435- 1746 UAUCUUAAAAGGGUGGUAG 1770 CUACCACCCUUUUAAGAUA 1453 XM_001103095577- 1747 CCAUGGAUUCUGGCGGUAU 1771 AUACCGCCAGAAUCCAUGG 595 XM_001103095790- 1748 UUAAUGAACUAAUUCGUUU 1772 AAACGAAUUAGUUCAUUAA 808 XM_0011030951479- 1749 AGGGUCAUUAGACAAAUGU 1773 ACAUUUGUCUAAUGACCCU 1497 XM_001103095794- 1750 UGAACUAAUUCGUUUUGAC 1774 GUCAAAACGAAUUAGUUCA 812 XM_0011030951150- 1751 UUCCCCAGCUGAUUAGUGU 1775 ACACUAAUCAGCUGGGGAA 1168 XM_0011030951473- 1752 UAUGUAAGGGUCAUUAGAC 1776 GUCUAAUGACCCUUACAUA 1491 *Target refers to target gene and location of target sequence. NM_001004210 is the gene for rat XBP-1. XM_001103095 is the sequence for Macaca mulatta (rhesus monkey) XBP-1.

TABLE 10 Target gene name and target sequence location for dsRNA targeting XBP-1 Duplex # Target gene and location of target sequence AD18027 NM_001004210_1128-1146 AD18028 NM_001004210_1129-1147 AD18029 NM_001004210_677-695 AD18030 NM_001004210_893-911 AD18031 NM_001004210_895-913 AD18032 NM_001004210_1127-1145 AD18033 NM_001004210_894-912 AD18034 NM_001004210_1760-1778 AD18035 NM_001004210_215-233 AD18036 NM_001004210_1759-1777 AD18037 NM_001004210_367-385 AD18038 NM_001004210_896-914 AD18039 NM_001004210_214-232 AD18040 NM_001004210_216-234 AD18041 XM_001103095_387-405 AD18042 XM_001103095_1151-1169 AD18043 XM_001103095_1466-1484 AD18044 XM_001103095_1435-1453 AD18045 XM_001103095_577-595 AD18046 XM_001103095_790-808 AD18047 XM_001103095_1479-1497 AD18048 XM_001103095_794-812 AD18049 XM_001103095_1150-1168 AD18050 XM_001103095_1473-1491 *Target refers to target gene and location of target sequence. NM_001004210 is the gene for rat XBP-1. XM_001103095 is the sequence for Macaca mulatta (rhesus monkey) XBP-1.

TABLE 11 Sequences of dsRNA targeting XBP-1, with Endolight chemistry modifications SEQ SEQ ID ID Duplex # NO Sense (5′-3′) NO Antisense (5′-3′) AD18027 4166 cccAGcuGAuuAGuGucuAdTsdT 1800 uAGAcACuAAUcAGCUGGGdTsdT AD18028 1777 ccAGcuGAuuAGuGucuAAdTsdT 1801 UuAGAcACuAAUcAGCUGGdTsdT AD18029 1778 cucccAGAGGucuAcccAGdTsdT 1802 CUGGGuAGACCUCUGGGAGdTsdT AD18030 1779 GAucAcccuGAAuucAuuGdTsdT 1803 cAAUGAAUUcAGGGUGAUCdTsdT AD18031 1780 ucAcccuGAAuucAuuGucdTsdT 1804 GAcAAUGAAUUcAGGGUGAdTsdT AD18032 1781 ccccAGcuGAuuAGuGucudTsdT 1805 AGAcACuAAUcAGCUGGGGdTsdT AD18033 1782 AucAcccuGAAuucAuuGudTsdT 1806 AcAAUGAAUUcAGGGUGAUdTsdT AD18034 1783 cAuuuAuuuAAAAcuAcccdTsdT 1807 GGGuAGUUUuAAAuAAAUGdTsdT AD18035 1784 AcuGAAAAAcAGAGuAGcAdTsdT 1808 UGCuACUCUGUUUUUcAGUdTsdT AD18036 1785 ccAuuuAuuuAAAAcuAccdTsdT 1809 GGuAGUUUuAAAuAAAUGGdTsdT AD18037 1786 uuGAGAAccAGGAGuuAAGdTsdT 1810 CUuAACUCCUGGUUCUcAAdTsdT AD18038 1787 cAcccuGAAuucAuuGucudTsdT 1811 AGAcAAUGAAUUcAGGGUGdTsdT AD18039 1788 AAcuGAAAAAcAGAGuAGcdTsdT 1812 GCuACUCUGUUUUUcAGUUdTsdT AD18040 1789 cuGAAAAAcAGAGuAGcAGdTsdT 1813 CUGCuACUCUGUUUUUcAGdTsdT AD18041 1790 AGAAAAucAGcuuuuAcGAdTsdT 1814 UCGuAAAAGCUGAUUUUCUdTsdT AD18042 1791 uccccAGcuGAuuAGuGucdTsdT 1815 GAcACuAAUcAGCUGGGGAdTsdT AD18043 1792 uAcuuAuuAuGuAAGGGucdTsdT 1816 GACCCUuAcAuAAuAAGuAdTsdT AD18044 1793 uAucuuAAAAGGGuGGuAGdTsdT 1817 CuACcACCCUUUuAAGAuAdTsdT AD18045 1794 ccAuGGAuucuGGcGGuAudTsdT 1818 AuACCGCcAGAAUCcAUGGdTsdT AD18046 1795 uuAAuGAAcuAAuucGuuudTsdT 1819 AAACGAAUuAGUUcAUuAAdTsdT AD18047 1796 AGGGucAuuAGAcAAAuGudTsdT 1820 AcAUUUGUCuAAUGACCCUdTsdT AD18048 1797 uGAAcuAAuucGuuuuGAcdTsdT 1821 GUcAAAACGAAUuAGUUcAdTsdT AD18049 1798 uuccccAGcuGAuuAGuGudTsdT 1822 AcACuAAUcAGCUGGGGAAdTsdT AD18050 1799 uAuGuAAGGGucAuuAGAcdTsdT 1823 GUCuAAUGACCCUuAcAuAdTsdT U, C, A, G: corresponding ribonucleotide; dT: deoxythymidine; u, c, a, g: corresponding 2′-O-methyl ribonucleotide; where nucleotides are written in sequence, they are connected by 3′-5′ phosphodiester groups; nucleotides with interjected “s” are connected by 3′-O-5′-O phosphorothiodiester groups.

TABLE 12 Sequences of dsRNA targeting both human and rhesus monkeyXBP-1. SEQ SEQ ID ID Target* sense (5′-3′) NO antisense (5′-3′) NO 100-118 CUGCUUCUGUCGGGGCAGCNN 1824 GCUGCCCCGACAGAAGCAGNN 28 1011-1029 GAGCUGGGUAUCUCAAAUCNN 1825 GAUUUGAGAUACCCAGCUCNN 28 101-119 UGCUUCUGUCGGGGCAGCCNN 1826 GGCUGCCCCGACAGAAGCANN 28 1012-1030 AGCUGGGUAUCUCAAAUCUNN 1827 AGAUUUGAGAUACCCAGCUNN 28 1013-1031 GCUGGGUAUCUCAAAUCUGNN 1828 CAGAUUUGAGAUACCCAGCNN 28 1014-1032 CUGGGUAUCUCAAAUCUGCNN 1829 GCAGAUUUGAGAUACCCAGNN 28 1015-1033 UGGGUAUCUCAAAUCUGCUNN 1830 AGCAGAUUUGAGAUACCCANN 28 1016-1034 GGGUAUCUCAAAUCUGCUUNN 1831 AAGCAGAUUUGAGAUACCCNN 28 1017-1035 GGUAUCUCAAAUCUGCUUUNN 1832 AAAGCAGAUUUGAGAUACCNN 28 1018-1036 GUAUCUCAAAUCUGCUUUCNN 1833 GAAAGCAGAUUUGAGAUACNN 28 1019-1037 UAUCUCAAAUCUGCUUUCANN 1834 UGAAAGCAGAUUUGAGAUANN 28 1020-1038 AUCUCAAAUCUGCUUUCAUNN 1835 AUGAAAGCAGAUUUGAGAUNN 28 1021-1039 UCUCAAAUCUGCUUUCAUCNN 1836 GAUGAAAGCAGAUUUGAGANN 28 102-120 GCUUCUGUCGGGGCAGCCCNN 1837 GGGCUGCCCCGACAGAAGCNN 28 1022-1040 CUCAAAUCUGCUUUCAUCCNN 1838 GGAUGAAAGCAGAUUUGAGNN 28 1023-1041 UCAAAUCUGCUUUCAUCCANN 1839 UGGAUGAAAGCAGAUUUGANN 28 1024-1042 CAAAUCUGCUUUCAUCCAGNN 1840 CUGGAUGAAAGCAGAUUUGNN 28 1025-1043 AAAUCUGCUUUCAUCCAGCNN 1841 GCUGGAUGAAAGCAGAUUUNN 29 1026-1044 AAUCUGCUUUCAUCCAGCCNN 1842 GGCUGGAUGAAAGCAGAUUNN 29 1027-1045 AUCUGCUUUCAUCCAGCCANN 1843 UGGCUGGAUGAAAGCAGAUNN 29 1028-1046 UCUGCUUUCAUCCAGCCACNN 1844 GUGGCUGGAUGAAAGCAGANN 29 1029-1047 CUGCUUUCAUCCAGCCACUNN 1845 AGUGGCUGGAUGAAAGCAGNN 29 1030-1048 UGCUUUCAUCCAGCCACUGNN 1846 CAGUGGCUGGAUGAAAGCANN 29 1031-1049 GCUUUCAUCCAGCCACUGCNN 1847 GCAGUGGCUGGAUGAAAGCNN 29 103-121 CUUCUGUCGGGGCAGCCCGNN 1848 CGGGCUGCCCCGACAGAAGNN 29 1032-1050 CUUUCAUCCAGCCACUGCCNN 1849 GGCAGUGGCUGGAUGAAAGNN 29 1033-1051 UUUCAUCCAGCCACUGCCCNN 1850 GGGCAGUGGCUGGAUGAAANN 29 104-122 UUCUGUCGGGGCAGCCCGCNN 1851 GCGGGCUGCCCCGACAGAANN 29 105-123 UCUGUCGGGGCAGCCCGCCNN 1852 GGCGGGCUGCCCCGACAGANN 29 1056-1074 CCAUCUUCCUGCCUACUGGNN 1853 CCAGUAGGCAGGAAGAUGGNN 29 1057-1075 CAUCUUCCUGCCUACUGGANN 1854 UCCAGUAGGCAGGAAGAUGNN 29 1058-1076 AUCUUCCUGCCUACUGGAUNN 1855 AUCCAGUAGGCAGGAAGAUNN 29 1059-1077 UCUUCCUGCCUACUGGAUGNN 1856 CAUCCAGUAGGCAGGAAGANN 29 1060-1078 CUUCCUGCCUACUGGAUGCNN 1857 GCAUCCAGUAGGCAGGAAGNN 29 1061-1079 UUCCUGCCUACUGGAUGCUNN 1858 AGCAUCCAGUAGGCAGGAANN 29 106-124 CUGUCGGGGCAGCCCGCCUNN 1859 AGGCGGGCUGCCCCGACAGNN 29 1062-1080 UCCUGCCUACUGGAUGCUUNN 1860 AAGCAUCCAGUAGGCAGGANN 29 1063-1081 CCUGCCUACUGGAUGCUUANN 1861 UAAGCAUCCAGUAGGCAGGNN 29 1064-1082 CUGCCUACUGGAUGCUUACNN 1862 GUAAGCAUCCAGUAGGCAGNN 29 1065-1083 UGCCUACUGGAUGCUUACANN 1863 UGUAAGCAUCCAGUAGGCANN 29 1066-1084 GCCUACUGGAUGCUUACAGNN 1864 CUGUAAGCAUCCAGUAGGCNN 29 1067-1085 CCUACUGGAUGCUUACAGUNN 1865 ACUGUAAGCAUCCAGUAGGNN 29 1068-1086 CUACUGGAUGCUUACAGUGNN 1866 CACUGUAAGCAUCCAGUAGNN 29 1069-1087 UACUGGAUGCUUACAGUGANN 1867 UCACUGUAAGCAUCCAGUANN 29 1070-1088 ACUGGAUGCUUACAGUGACNN 1868 GUCACUGUAAGCAUCCAGUNN 29 1071-1089 CUGGAUGCUUACAGUGACUNN 1869 AGUCACUGUAAGCAUCCAGNN 29 107-125 UGUCGGGGCAGCCCGCCUCNN 1870 GAGGCGGGCUGCCCCGACANN 29 1072-1090 UGGAUGCUUACAGUGACUGNN 1871 CAGUCACUGUAAGCAUCCANN 29 1073-1091 GGAUGCUUACAGUGACUGUNN 1872 ACAGUCACUGUAAGCAUCCNN 29 1074-1092 GAUGCUUACAGUGACUGUGNN 1873 CACAGUCACUGUAAGCAUCNN 29 1075-1093 AUGCUUACAGUGACUGUGGNN 1874 CCACAGUCACUGUAAGCAUNN 29 1076-1094 UGCUUACAGUGACUGUGGANN 1875 UCCACAGUCACUGUAAGCANN 29 1077-1095 GCUUACAGUGACUGUGGAUNN 1876 AUCCACAGUCACUGUAAGCNN 29 1078-1096 CUUACAGUGACUGUGGAUANN 1877 UAUCCACAGUCACUGUAAGNN 29 108-126 GUCGGGGCAGCCCGCCUCCNN 1878 GGAGGCGGGCUGCCCCGACNN 29 109-127 UCGGGGCAGCCCGCCUCCGNN 1879 CGGAGGCGGGCUGCCCCGANN 29 110-128 CGGGGCAGCCCGCCUCCGCNN 1880 GCGGAGGCGGGCUGCCCCGNN 29 111-129 GGGGCAGCCCGCCUCCGCCNN 1881 GGCGGAGGCGGGCUGCCCCNN 29 1116-1134 UUCAGUGACAUGUCCUCUCNN 1882 GAGAGGACAUGUCACUGAANN 29 112-130 GGGCAGCCCGCCUCCGCCGNN 1883 CGGCGGAGGCGGGCUGCCCNN 29 113-131 GGCAGCCCGCCUCCGCCGCNN 1884 GCGGCGGAGGCGGGCUGCCNN 29 1136-1154 GCUUGGUGUAAACCAUUCUNN 1885 AGAAUGGUUUACACCAAGCNN 29 1137-1155 CUUGGUGUAAACCAUUCUUNN 1886 AAGAAUGGUUUACACCAAGNN 29 1138-1156 UUGGUGUAAACCAUUCUUGNN 1887 CAAGAAUGGUUUACACCAANN 29 1139-1157 UGGUGUAAACCAUUCUUGGNN 1888 CCAAGAAUGGUUUACACCANN 29 1140-1158 GGUGUAAACCAUUCUUGGGNN 1889 CCCAAGAAUGGUUUACACCNN 29 1141-1159 GUGUAAACCAUUCUUGGGANN 1890 UCCCAAGAAUGGUUUACACNN 29 114-132 GCAGCCCGCCUCCGCCGCCNN 1891 GGCGGCGGAGGCGGGCUGCNN 29 1142-1160 UGUAAACCAUUCUUGGGAGNN 1892 CUCCCAAGAAUGGUUUACANN 29 1143-1161 GUAAACCAUUCUUGGGAGGNN 1893 CCUCCCAAGAAUGGUUUACNN 29 1144-1162 UAAACCAUUCUUGGGAGGANN 1894 UCCUCCCAAGAAUGGUUUANN 29 1145-1163 AAACCAUUCUUGGGAGGACNN 1895 GUCCUCCCAAGAAUGGUUUNN 29 1146-1164 AACCAUUCUUGGGAGGACANN 1896 UGUCCUCCCAAGAAUGGUUNN 29 1147-1165 ACCAUUCUUGGGAGGACACNN 1897 GUGUCCUCCCAAGAAUGGUNN 29 1148-1166 CCAUUCUUGGGAGGACACUNN 1898 AGUGUCCUCCCAAGAAUGGNN 29 1149-1167 CAUUCUUGGGAGGACACUUNN 1899 AAGUGUCCUCCCAAGAAUGNN 29 1150-1168 AUUCUUGGGAGGACACUUUNN 1900 AAAGUGUCCUCCCAAGAAUNN 29 1151-1169 UUCUUGGGAGGACACUUUUNN 1901 AAAAGUGUCCUCCCAAGAANN 29 115-133 CAGCCCGCCUCCGCCGCCGNN 1902 CGGCGGCGGAGGCGGGCUGNN 29 1152-1170 UCUUGGGAGGACACUUUUGNN 1903 CAAAAGUGUCCUCCCAAGANN 29 1153-1171 CUUGGGAGGACACUUUUGCNN 1904 GCAAAAGUGUCCUCCCAAGNN 29 1154-1172 UUGGGAGGACACUUUUGCCNN 1905 GGCAAAAGUGUCCUCCCAANN 29 1155-1173 UGGGAGGACACUUUUGCCANN 1906 UGGCAAAAGUGUCCUCCCANN 29 1156-1174 GGGAGGACACUUUUGCCAANN 1907 UUGGCAAAAGUGUCCUCCCNN 29 1157-1175 GGAGGACACUUUUGCCAAUNN 1908 AUUGGCAAAAGUGUCCUCCNN 29 1158-1176 GAGGACACUUUUGCCAAUGNN 1909 CAUUGGCAAAAGUGUCCUCNN 29 1159-1177 AGGACACUUUUGCCAAUGANN 1910 UCAUUGGCAAAAGUGUCCUNN 29 1160-1178 GGACACUUUUGCCAAUGAANN 1911 UUCAUUGGCAAAAGUGUCCNN 29 1161-1179 GACACUUUUGCCAAUGAACNN 1912 GUUCAUUGGCAAAAGUGUCNN 29 116-134 AGCCCGCCUCCGCCGCCGGNN 1913 CCGGCGGCGGAGGCGGGCUNN 29 1162-1180 ACACUUUUGCCAAUGAACUNN 1914 AGUUCAUUGGCAAAAGUGUNN 29 117-135 GCCCGCCUCCGCCGCCGGANN 1915 UCCGGCGGCGGAGGCGGGCNN 29 118-136 CCCGCCUCCGCCGCCGGAGNN 1916 CUCCGGCGGCGGAGGCGGGNN 29 1182-1200 UUUCCCCAGCUGAUUAGUGNN 1917 CACUAAUCAGCUGGGGAAANN 29 1183-1201 UUCCCCAGCUGAUUAGUGUNN 1918 ACACUAAUCAGCUGGGGAANN 29 1184-1202 UCCCCAGCUGAUUAGUGUCNN 1919 GACACUAAUCAGCUGGGGANN 29 1185-1203 CCCCAGCUGAUUAGUGUCUNN 1920 AGACACUAAUCAGCUGGGGNN 29 1186-1204 CCCAGCUGAUUAGUGUCUANN 1921 UAGACACUAAUCAGCUGGGNN 29 1187-1205 CCAGCUGAUUAGUGUCUAANN 1922 UUAGACACUAAUCAGCUGGNN 29 1188-1206 CAGCUGAUUAGUGUCUAAGNN 1923 CUUAGACACUAAUCAGCUGNN 29 1189-1207 AGCUGAUUAGUGUCUAAGGNN 1924 CCUUAGACACUAAUCAGCUNN 29 1190-1208 GCUGAUUAGUGUCUAAGGANN 1925 UCCUUAGACACUAAUCAGCNN 29 1191-1209 CUGAUUAGUGUCUAAGGAANN 1926 UUCCUUAGACACUAAUCAGNN 29 119-137 CCGCCUCCGCCGCCGGAGCNN 1927 GCUCCGGCGGCGGAGGCGGNN 29 1192-1210 UGAUUAGUGUCUAAGGAAUNN 1928 AUUCCUUAGACACUAAUCANN 29 1193-1211 GAUUAGUGUCUAAGGAAUGNN 1929 CAUUCCUUAGACACUAAUCNN 29 1194-1212 AUUAGUGUCUAAGGAAUGANN 1930 UCAUUCCUUAGACACUAAUNN 29 1195-1213 UUAGUGUCUAAGGAAUGAUNN 1931 AUCAUUCCUUAGACACUAANN 29 1196-1214 UAGUGUCUAAGGAAUGAUCNN 1932 GAUCAUUCCUUAGACACUANN 29 1197-1215 AGUGUCUAAGGAAUGAUCCNN 1933 GGAUCAUUCCUUAGACACUNN 29 1198-1216 GUGUCUAAGGAAUGAUCCANN 1934 UGGAUCAUUCCUUAGACACNN 29 120-138 CGCCUCCGCCGCCGGAGCCNN 1935 GGCUCCGGCGGCGGAGGCGNN 29 121-139 GCCUCCGCCGCCGGAGCCCNN 1936 GGGCUCCGGCGGCGGAGGCNN 29 1218-1236 UACUGUUGCCCUUUUCCUUNN 1937 AAGGAAAAGGGCAACAGUANN 29 1219-1237 ACUGUUGCCCUUUUCCUUGNN 1938 CAAGGAAAAGGGCAACAGUNN 29 1220-1238 CUGUUGCCCUUUUCCUUGANN 1939 UCAAGGAAAAGGGCAACAGNN 29 1221-1239 UGUUGCCCUUUUCCUUGACNN 1940 GUCAAGGAAAAGGGCAACANN 29 122-140 CCUCCGCCGCCGGAGCCCCNN 1941 GGGGCUCCGGCGGCGGAGGNN 30 1222-1240 GUUGCCCUUUUCCUUGACUNN 1942 AGUCAAGGAAAAGGGCAACNN 30 1223-1241 UUGCCCUUUUCCUUGACUANN 1943 UAGUCAAGGAAAAGGGCAANN 30 1224-1242 UGCCCUUUUCCUUGACUAUNN 1944 AUAGUCAAGGAAAAGGGCANN 30 1225-1243 GCCCUUUUCCUUGACUAUUNN 1945 AAUAGUCAAGGAAAAGGGCNN 30 1226-1244 CCCUUUUCCUUGACUAUUANN 1946 UAAUAGUCAAGGAAAAGGGNN 30 1227-1245 CCUUUUCCUUGACUAUUACNN 1947 GUAAUAGUCAAGGAAAAGGNN 30 1228-1246 CUUUUCCUUGACUAUUACANN 1948 UGUAAUAGUCAAGGAAAAGNN 30 1229-1247 UUUUCCUUGACUAUUACACNN 1949 GUGUAAUAGUCAAGGAAAANN 30 1230-1248 UUUCCUUGACUAUUACACUNN 1950 AGUGUAAUAGUCAAGGAAANN 30 1231-1249 UUCCUUGACUAUUACACUGNN 1951 CAGUGUAAUAGUCAAGGAANN 30 123-141 CUCCGCCGCCGGAGCCCCGNN 1952 CGGGGCUCCGGCGGCGGAGNN 30 1232-1250 UCCUUGACUAUUACACUGCNN 1953 GCAGUGUAAUAGUCAAGGANN 30 1233-1251 CCUUGACUAUUACACUGCCNN 1954 GGCAGUGUAAUAGUCAAGGNN 30 1234-1252 CUUGACUAUUACACUGCCUNN 1955 AGGCAGUGUAAUAGUCAAGNN 30 1235-1253 UUGACUAUUACACUGCCUGNN 1956 CAGGCAGUGUAAUAGUCAANN 30 1236-1254 UGACUAUUACACUGCCUGGNN 1957 CCAGGCAGUGUAAUAGUCANN 30 1237-1255 GACUAUUACACUGCCUGGANN 1958 UCCAGGCAGUGUAAUAGUCNN 30 1238-1256 ACUAUUACACUGCCUGGAGNN 1959 CUCCAGGCAGUGUAAUAGUNN 30 1239-1257 CUAUUACACUGCCUGGAGGNN 1960 CCUCCAGGCAGUGUAAUAGNN 30 1240-1258 UAUUACACUGCCUGGAGGANN 1961 UCCUCCAGGCAGUGUAAUANN 30 1241-1259 AUUACACUGCCUGGAGGAUNN 1962 AUCCUCCAGGCAGUGUAAUNN 30 124-142 UCCGCCGCCGGAGCCCCGGNN 1963 CCGGGGCUCCGGCGGCGGANN 30 1242-1260 UUACACUGCCUGGAGGAUANN 1964 UAUCCUCCAGGCAGUGUAANN 30 1243-1261 UACACUGCCUGGAGGAUAGNN 1965 CUAUCCUCCAGGCAGUGUANN 30 1244-1262 ACACUGCCUGGAGGAUAGCNN 1966 GCUAUCCUCCAGGCAGUGUNN 30 1245-1263 CACUGCCUGGAGGAUAGCANN 1967 UGCUAUCCUCCAGGCAGUGNN 30 1246-1264 ACUGCCUGGAGGAUAGCAGNN 1968 CUGCUAUCCUCCAGGCAGUNN 30 125-143 CCGCCGCCGGAGCCCCGGCNN 1969 GCCGGGGCTCCGGCGGCGGNN 30 126-144 CGCCGCCGGAGCCCCGGCCNN 1970 GGCCGGGGCTCCGGCGGCGNN 30 127-145 GCCGCCGGAGCCCCGGCCGNN 1971 CGGCCGGGGCTCCGGCGGCNN 30 1280-1298 CUUCAUUCAAAAAGCCAAANN 1972 UUUGGCUUUUUGAAUGAAGNN 30 1281-1299 UUCAUUCAAAAAGCCAAAANN 1973 UUUUGGCUUUUUGAAUGAANN 30 128-146 CCGCCGGAGCCCCGGCCGGNN 1974 CCGGCCGGGGCTCCGGCGGNN 30 1282-1300 UCAUUCAAAAAGCCAAAAUNN 1975 AUUUUGGCUUUUUGAAUGANN 30 1283-1301 CAUUCAAAAAGCCAAAAUANN 1976 UAUUUUGGCUUUUUGAAUGNN 30 1284-1302 AUUCAAAAAGCCAAAAUAGNN 1977 CUAUUUUGGCUUUUUGAAUNN 30 1285-1303 UUCAAAAAGCCAAAAUAGANN 1978 UCUAUUUUGGCUUUUUGAANN 30 1286-1304 UCAAAAAGCCAAAAUAGAGNN 1979 CUCUAUUUUGGCUUUUUGANN 30 1287-1305 CAAAAAGCCAAAAUAGAGANN 1980 UCUCUAUUUUGGCUUUUUGNN 30 1288-1306 AAAAAGCCAAAAUAGAGAGNN 1981 CUCUCUAUUUUGGCUUUUUNN 30 1289-1307 AAAAGCCAAAAUAGAGAGUNN 1982 ACUCUCUAUUUUGGCUUUUNN 30 1290-1308 AAAGCCAAAAUAGAGAGUANN 1983 UACUCUCUAUUUUGGCUUUNN 30 129-147 CGCCGGAGCCCCGGCCGGCNN 1984 GCCGGCCGGGGCTCCGGCGNN 30 130-148 GCCGGAGCCCCGGCCGGCCNN 1985 GGCCGGCCGGGGCTCCGGCNN 30 1310-1328 ACAGUCCUAGAGAAUUCCUNN 1986 AGGAAUUCUCUAGGACUGUNN 30 131-149 CCGGAGCCCCGGCCGGCCANN 1987 TGGCCGGCCGGGGCTCCGGNN 30 132-150 CGGAGCCCCGGCCGGCCAGNN 1988 CTGGCCGGCCGGGGCTCCGNN 30 1330-1348 UAUUUGUUCAGAUCUCAUANN 1989 UAUGAGAUCUGAACAAAUANN 30 1331-1349 AUUUGUUCAGAUCUCAUAGNN 1990 CUAUGAGAUCUGAACAAAUNN 30 133-151 GGAGCCCCGGCCGGCCAGGNN 1991 CCTGGCCGGCCGGGGCTCCNN 30 1332-1350 UUUGUUCAGAUCUCAUAGANN 1992 UCUAUGAGAUCUGAACAAANN 30 1333-1351 UUGUUCAGAUCUCAUAGAUNN 1993 AUCUAUGAGAUCUGAACAANN 30 1334-1352 UGUUCAGAUCUCAUAGAUGNN 1994 CAUCUAUGAGAUCUGAACANN 30 1335-1353 GUUCAGAUCUCAUAGAUGANN 1995 UCAUCUAUGAGAUCUGAACNN 30 134-152 GAGCCCCGGCCGGCCAGGCNN 1996 GCCTGGCCGGCCGGGGCTCNN 30 135-153 AGCCCCGGCCGGCCAGGCCNN 1997 GGCCTGGCCGGCCGGGGCTNN 30 136-154 GCCCCGGCCGGCCAGGCCCNN 1998 GGGCCTGGCCGGCCGGGGCNN 30 1365-1383 UGUCUUUUGACAUCCAGCANN 1999 UGCUGGAUGUCAAAAGACANN 30 1366-1384 GUCUUUUGACAUCCAGCAGNN 2000 CUGCUGGAUGUCAAAAGACNN 30 1367-1385 UCUUUUGACAUCCAGCAGUNN 2001 ACUGCUGGAUGUCAAAAGANN 30 1368-1386 CUUUUGACAUCCAGCAGUCNN 2002 GACUGCUGGAUGUCAAAAGNN 30 1369-1387 UUUUGACAUCCAGCAGUCCNN 2003 GGACUGCUGGAUGUCAAAANN 30 1370-1388 UUUGACAUCCAGCAGUCCANN 2004 UGGACUGCUGGAUGUCAAANN 30 1371-1389 UUGACAUCCAGCAGUCCAANN 2005 UUGGACUGCUGGAUGUCAANN 30 137-155 CCCCGGCCGGCCAGGCCCUNN 2006 AGGGCCUGGCCGGCCGGGGNN 30 138-156 CCCGGCCGGCCAGGCCCUGNN 2007 CAGGGCCUGGCCGGCCGGGNN 30 1391-1409 GUAUUGAGACAUAUUACUGNN 2008 CAGUAAUAUGUCUCAAUACNN 30 139-157 CCGGCCGGCCAGGCCCUGCNN 2009 GCAGGGCCUGGCCGGCCGGNN 30 140-158 CGGCCGGCCAGGCCCUGCCNN 2010 GGCAGGGCCUGGCCGGCCGNN 30 141-159 GGCCGGCCAGGCCCUGCCGNN 2011 CGGCAGGGCCUGGCCGGCCNN 30 1414-1432 UAAGAAAUAUUACUAUAAUNN 2012 AUUAUAGUAAUAUUUCUUANN 30 1415-1433 AAGAAAUAUUACUAUAAUUNN 2013 AAUUAUAGUAAUAUUUCUUNN 30 1416-1434 AGAAAUAUUACUAUAAUUGNN 2014 CAAUUAUAGUAAUAUUUCUNN 30 1417-1435 GAAAUAUUACUAUAAUUGANN 2015 UCAAUUAUAGUAAUAUUUCNN 30 1418-1436 AAAUAUUACUAUAAUUGAGNN 2016 CUCAAUUAUAGUAAUAUUUNN 30 1419-1437 AAUAUUACUAUAAUUGAGANN 2017 UCUCAAUUAUAGUAAUAUUNN 30 1420-1438 AUAUUACUAUAAUUGAGAANN 2018 UUCUCAAUUAUAGUAAUAUNN 30 1421-1439 UAUUACUAUAAUUGAGAACNN 2019 GUUCUCAAUUAUAGUAAUANN 30 142-160 GCCGGCCAGGCCCUGCCGCNN 2020 GCGGCAGGGCCUGGCCGGCNN 30 1422-1440 AUUACUAUAAUUGAGAACUNN 2021 AGUUCUCAAUUAUAGUAAUNN 30 1423-1441 UUACUAUAAUUGAGAACUANN 2022 UAGUUCUCAAUUAUAGUAANN 30 1424-1442 UACUAUAAUUGAGAACUACNN 2023 GUAGUUCUCAAUUAUAGUANN 30 1425-1443 ACUAUAAUUGAGAACUACANN 2024 UGUAGUUCUCAAUUAUAGUNN 30 1426-1444 CUAUAAUUGAGAACUACAGNN 2025 CUGUAGUUCUCAAUUAUAGNN 30 1427-1445 UAUAAUUGAGAACUACAGCNN 2026 GCUGUAGUUCUCAAUUAUANN 30 1428-1446 AUAAUUGAGAACUACAGCUNN 2027 AGCUGUAGUUCUCAAUUAUNN 30 1429-1447 UAAUUGAGAACUACAGCUUNN 2028 AAGCUGUAGUUCUCAAUUANN 30 1430-1448 AAUUGAGAACUACAGCUUUNN 2029 AAAGCUGUAGUUCUCAAUUNN 30 1431-1449 AUUGAGAACUACAGCUUUUNN 2030 AAAAGCUGUAGUUCUCAAUNN 30 143-161 CCGGCCAGGCCCUGCCGCUNN 2031 AGCGGCAGGGCCUGGCCGGNN 30 1432-1450 UUGAGAACUACAGCUUUUANN 2032 UAAAAGCUGUAGUUCUCAANN 30 1433-1451 UGAGAACUACAGCUUUUAANN 2033 UUAAAAGCUGUAGUUCUCANN 30 1434-1452 GAGAACUACAGCUUUUAAGNN 2034 CUUAAAAGCUGUAGUUCUCNN 30 1435-1453 AGAACUACAGCUUUUAAGANN 2035 UCUUAAAAGCUGUAGUUCUNN 30 1436-1454 GAACUACAGCUUUUAAGAUNN 2036 AUCUUAAAAGCUGUAGUUCNN 30 1437-1455 AACUACAGCUUUUAAGAUUNN 2037 AAUCUUAAAAGCUGUAGUUNN 30 1438-1456 ACUACAGCUUUUAAGAUUGNN 2038 CAAUCUUAAAAGCUGUAGUNN 30 1439-1457 CUACAGCUUUUAAGAUUGUNN 2039 ACAAUCUUAAAAGCUGUAGNN 30 1440-1458 UACAGCUUUUAAGAUUGUANN 2040 UACAAUCUUAAAAGCUGUANN 30 1441-1459 ACAGCUUUUAAGAUUGUACNN 2041 GUACAAUCUUAAAAGCUGUNN 31 144-162 CGGCCAGGCCCUGCCGCUCNN 2042 GAGCGGCAGGGCCUGGCCGNN 31 1442-1460 CAGCUUUUAAGAUUGUACUNN 2043 AGUACAAUCUUAAAAGCUGNN 31 1443-1461 AGCUUUUAAGAUUGUACUUNN 2044 AAGUACAAUCUUAAAAGCUNN 31 1444-1462 GCUUUUAAGAUUGUACUUUNN 2045 AAAGUACAAUCUUAAAAGCNN 31 1445-1463 CUUUUAAGAUUGUACUUUUNN 2046 AAAAGUACAAUCUUAAAAGNN 31 1446-1464 UUUUAAGAUUGUACUUUUANN 2047 UAAAAGUACAAUCUUAAAANN 31 1447-1465 UUUAAGAUUGUACUUUUAUNN 2048 AUAAAAGUACAAUCUUAAANN 31 1448-1466 UUAAGAUUGUACUUUUAUCNN 2049 GAUAAAAGUACAAUCUUAANN 31 1449-1467 UAAGAUUGUACUUUUAUCUNN 2050 AGAUAAAAGUACAAUCUUANN 31 1450-1468 AAGAUUGUACUUUUAUCUUNN 2051 AAGAUAAAAGUACAAUCUUNN 31 1451-1469 AGAUUGUACUUUUAUCUUANN 2052 UAAGAUAAAAGUACAAUCUNN 31 145-163 GGCCAGGCCCUGCCGCUCANN 2053 UGAGCGGCAGGGCCUGGCCNN 31 1452-1470 GAUUGUACUUUUAUCUUAANN 2054 UUAAGAUAAAAGUACAAUCNN 31 1453-1471 AUUGUACUUUUAUCUUAAANN 2055 UUUAAGAUAAAAGUACAAUNN 31 1454-1472 UUGUACUUUUAUCUUAAAANN 2056 UUUUAAGAUAAAAGUACAANN 31 1455-1473 UGUACUUUUAUCUUAAAAGNN 2057 CUUUUAAGAUAAAAGUACANN 31 1456-1474 GUACUUUUAUCUUAAAAGGNN 2058 CCUUUUAAGAUAAAAGUACNN 31 1457-1475 UACUUUUAUCUUAAAAGGGNN 2059 CCCUUUUAAGAUAAAAGUANN 31 1458-1476 ACUUUUAUCUUAAAAGGGUNN 2060 ACCCUUUUAAGAUAAAAGUNN 31 1459-1477 CUUUUAUCUUAAAAGGGUGNN 2061 CACCCUUUUAAGAUAAAAGNN 31 1460-1478 UUUUAUCUUAAAAGGGUGGNN 2062 CCACCCUUUUAAGAUAAAANN 31 1461-1479 UUUAUCUUAAAAGGGUGGUNN 2063 ACCACCCUUUUAAGAUAAANN 31 146-164 GCCAGGCCCUGCCGCUCAUNN 2064 AUGAGCGGCAGGGCCUGGCNN 31 1462-1480 UUAUCUUAAAAGGGUGGUANN 2065 UACCACCCUUUUAAGAUAANN 31 1463-1481 UAUCUUAAAAGGGUGGUAGNN 2066 CUACCACCCUUUUAAGAUANN 31 1464-1482 AUCUUAAAAGGGUGGUAGUNN 2067 ACUACCACCCUUUUAAGAUNN 31 1465-1483 UCUUAAAAGGGUGGUAGUUNN 2068 AACUACCACCCUUUUAAGANN 31 1466-1484 CUUAAAAGGGUGGUAGUUUNN 2069 AAACUACCACCCUUUUAAGNN 31 147-165 CCAGGCCCUGCCGCUCAUGNN 2070 CAUGAGCGGCAGGGCCUGGNN 31 148-166 CAGGCCCUGCCGCUCAUGGNN 2071 CCAUGAGCGGCAGGGCCUGNN 31 1486-1504 CCCUAAAAUACUUAUUAUGNN 2072 CAUAAUAAGUAUUUUAGGGNN 31 1487-1505 CCUAAAAUACUUAUUAUGUNN 2073 ACAUAAUAAGUAUUUUAGGNN 31 1488-1506 CUAAAAUACUUAUUAUGUANN 2074 UACAUAAUAAGUAUUUUAGNN 31 1489-1507 UAAAAUACUUAUUAUGUAANN 2075 UUACAUAAUAAGUAUUUUANN 31 1490-1508 AAAAUACUUAUUAUGUAAGNN 2076 CUUACAUAAUAAGUAUUUUNN 31 1491-1509 AAAUACUUAUUAUGUAAGGNN 2077 CCUUACAUAAUAAGUAUUUNN 31 149-167 AGGCCCUGCCGCUCAUGGUNN 2078 ACCAUGAGCGGCAGGGCCUNN 31 1492-1510 AAUACUUAUUAUGUAAGGGNN 2079 CCCUUACAUAAUAAGUAUUNN 31 1493-1511 AUACUUAUUAUGUAAGGGUNN 2080 ACCCUUACAUAAUAAGUAUNN 31 1494-1512 UACUUAUUAUGUAAGGGUCNN 2081 GACCCUUACAUAAUAAGUANN 31 1495-1513 ACUUAUUAUGUAAGGGUCANN 2082 UGACCCUUACAUAAUAAGUNN 31 1496-1514 CUUAUUAUGUAAGGGUCAUNN 2083 AUGACCCUUACAUAAUAAGNN 31 1497-1515 UUAUUAUGUAAGGGUCAUUNN 2084 AAUGACCCUUACAUAAUAANN 31 1498-1516 UAUUAUGUAAGGGUCAUUANN 2085 UAAUGACCCUUACAUAAUANN 31 1499-1517 AUUAUGUAAGGGUCAUUAGNN 2086 CUAAUGACCCUUACAUAAUNN 31 1500-1518 UUAUGUAAGGGUCAUUAGANN 2087 UCUAAUGACCCUUACAUAANN 31 1501-1519 UAUGUAAGGGUCAUUAGACNN 2088 GUCUAAUGACCCUUACAUANN 31 150-168 GGCCCUGCCGCUCAUGGUGNN 2089 CACCAUGAGCGGCAGGGCCNN 31 1502-1520 AUGUAAGGGUCAUUAGACANN 2090 UGUCUAAUGACCCUUACAUNN 31 1503-1521 UGUAAGGGUCAUUAGACAANN 2091 UUGUCUAAUGACCCUUACANN 31 1504-1522 GUAAGGGUCAUUAGACAAANN 2092 UUUGUCUAAUGACCCUUACNN 31 1505-1523 UAAGGGUCAUUAGACAAAUNN 2093 AUUUGUCUAAUGACCCUUANN 31 1506-1524 AAGGGUCAUUAGACAAAUGNN 2094 CAUUUGUCUAAUGACCCUUNN 31 1507-1525 AGGGUCAUUAGACAAAUGUNN 2095 ACAUUUGUCUAAUGACCCUNN 31 1508-1526 GGGUCAUUAGACAAAUGUCNN 2096 GACAUUUGUCUAAUGACCCNN 31 1509-1527 GGUCAUUAGACAAAUGUCUNN 2097 AGACAUUUGUCUAAUGACCNN 31 1510-1528 GUCAUUAGACAAAUGUCUUNN 2098 AAGACAUUUGUCUAAUGACNN 31 1511-1529 UCAUUAGACAAAUGUCUUGNN 2099 CAAGACAUUUGUCUAAUGANN 31 151-169 GCCCUGCCGCUCAUGGUGCNN 2100 GCACCAUGAGCGGCAGGGCNN 31 1512-1530 CAUUAGACAAAUGUCUUGANN 2101 UCAAGACAUUUGUCUAAUGNN 31 1513-1531 AUUAGACAAAUGUCUUGAANN 2102 UUCAAGACAUUUGUCUAAUNN 31 1514-1532 UUAGACAAAUGUCUUGAAGNN 2103 CUUCAAGACAUUUGUCUAANN 31 1515-1533 UAGACAAAUGUCUUGAAGUNN 2104 ACUUCAAGACAUUUGUCUANN 31 1516-1534 AGACAAAUGUCUUGAAGUANN 2105 UACUUCAAGACAUUUGUCUNN 31 1517-1535 GACAAAUGUCUUGAAGUAGNN 2106 CUACUUCAAGACAUUUGUCNN 31 1518-1536 ACAAAUGUCUUGAAGUAGANN 2107 UCUACUUCAAGACAUUUGUNN 31 152-170 CCCUGCCGCUCAUGGUGCCNN 2108 GGCACCAUGAGCGGCAGGGNN 31 153-171 CCUGCCGCUCAUGGUGCCANN 2109 UGGCACCAUGAGCGGCAGGNN 31 1541-1559 GAAUUUAUGAAUGGUUCUUNN 2110 AAGAACCAUUCAUAAAUUCNN 31 154-172 CUGCCGCUCAUGGUGCCAGNN 2111 CUGGCACCAUGAGCGGCAGNN 31 1542-1560 AAUUUAUGAAUGGUUCUUUNN 2112 AAAGAACCAUUCAUAAAUUNN 31 1543-1561 AUUUAUGAAUGGUUCUUUANN 2113 UAAAGAACCAUUCAUAAAUNN 31 1544-1562 UUUAUGAAUGGUUCUUUAUNN 2114 AUAAAGAACCAUUCAUAAANN 31 1545-1563 UUAUGAAUGGUUCUUUAUCNN 2115 GAUAAAGAACCAUUCAUAANN 31 1546-1564 UAUGAAUGGUUCUUUAUCANN 2116 UGAUAAAGAACCAUUCAUANN 31 1547-1565 AUGAAUGGUUCUUUAUCAUNN 2117 AUGAUAAAGAACCAUUCAUNN 31 1548-1566 UGAAUGGUUCUUUAUCAUUNN 2118 AAUGAUAAAGAACCAUUCANN 31 1549-1567 GAAUGGUUCUUUAUCAUUUNN 2119 AAAUGAUAAAGAACCAUUCNN 31 1550-1568 AAUGGUUCUUUAUCAUUUCNN 2120 GAAAUGAUAAAGAACCAUUNN 31 1551-1569 AUGGUUCUUUAUCAUUUCUNN 2121 AGAAAUGAUAAAGAACCAUNN 31 155-173 UGCCGCUCAUGGUGCCAGCNN 2122 GCUGGCACCAUGAGCGGCANN 31 1552-1570 UGGUUCUUUAUCAUUUCUCNN 2123 GAGAAAUGAUAAAGAACCANN 31 1553-1571 GGUUCUUUAUCAUUUCUCUNN 2124 AGAGAAAUGAUAAAGAACCNN 31 1554-1572 GUUCUUUAUCAUUUCUCUUNN 2125 AAGAGAAAUGAUAAAGAACNN 31 1555-1573 UUCUUUAUCAUUUCUCUUCNN 2126 GAAGAGAAAUGAUAAAGAANN 31 1556-1574 UCUUUAUCAUUUCUCUUCCNN 2127 GGAAGAGAAAUGAUAAAGANN 31 1557-1575 CUUUAUCAUUUCUCUUCCCNN 2128 GGGAAGAGAAAUGAUAAAGNN 31 1558-1576 UUUAUCAUUUCUCUUCCCCNN 2129 GGGGAAGAGAAAUGAUAAANN 31 1559-1577 UUAUCAUUUCUCUUCCCCCNN 2130 GGGGGAAGAGAAAUGAUAANN 31 1560-1578 UAUCAUUUCUCUUCCCCCUNN 2131 AGGGGGAAGAGAAAUGAUANN 31 1561-1579 AUCAUUUCUCUUCCCCCUUNN 2132 AAGGGGGAAGAGAAAUGAUNN 31 156-174 GCCGCUCAUGGUGCCAGCCNN 2133 GGCUGGCACCAUGAGCGGCNN 31 1562-1580 UCAUUUCUCUUCCCCCUUUNN 2134 AAAGGGGGAAGAGAAAUGANN 31 1563-1581 CAUUUCUCUUCCCCCUUUUNN 2135 AAAAGGGGGAAGAGAAAUGNN 31 1564-1582 AUUUCUCUUCCCCCUUUUUNN 2136 AAAAAGGGGGAAGAGAAAUNN 31 1565-1583 UUUCUCUUCCCCCUUUUUGNN 2137 CAAAAAGGGGGAAGAGAAANN 31 1566-1584 UUCUCUUCCCCCUUUUUGGNN 2138 CCAAAAAGGGGGAAGAGAANN 31 1567-1585 UCUCUUCCCCCUUUUUGGCNN 2139 GCCAAAAAGGGGGAAGAGANN 31 1568-1586 CUCUUCCCCCUUUUUGGCANN 2140 UGCCAAAAAGGGGGAAGAGNN 31 1569-1587 UCUUCCCCCUUUUUGGCAUNN 2141 AUGCCAAAAAGGGGGAAGANN 32 1570-1588 CUUCCCCCUUUUUGGCAUCNN 2142 GAUGCCAAAAAGGGGGAAGNN 32 1571-1589 UUCCCCCUUUUUGGCAUCCNN 2143 GGAUGCCAAAAAGGGGGAANN 32 157-175 CCGCUCAUGGUGCCAGCCCNN 2144 GGGCUGGCACCAUGAGCGGNN 32 1572-1590 UCCCCCUUUUUGGCAUCCUNN 2145 AGGAUGCCAAAAAGGGGGANN 32 1573-1591 CCCCCUUUUUGGCAUCCUGNN 2146 CAGGAUGCCAAAAAGGGGGNN 32 1574-1592 CCCCUUUUUGGCAUCCUGGNN 2147 CCAGGAUGCCAAAAAGGGGNN 32 1575-1593 CCCUUUUUGGCAUCCUGGCNN 2148 GCCAGGAUGCCAAAAAGGGNN 32 1576-1594 CCUUUUUGGCAUCCUGGCUNN 2149 AGCCAGGAUGCCAAAAAGGNN 32 1577-1595 CUUUUUGGCAUCCUGGCUUNN 2150 AAGCCAGGAUGCCAAAAAGNN 32 1578-1596 UUUUUGGCAUCCUGGCUUGNN 2151 CAAGCCAGGAUGCCAAAAANN 32 1579-1597 UUUUGGCAUCCUGGCUUGCNN 2152 GCAAGCCAGGAUGCCAAAANN 32 1580-1598 UUUGGCAUCCUGGCUUGCCNN 2153 GGCAAGCCAGGAUGCCAAANN 32 1581-1599 UUGGCAUCCUGGCUUGCCUNN 2154 AGGCAAGCCAGGAUGCCAANN 32 158-176 CGCUCAUGGUGCCAGCCCANN 2155 UGGGCUGGCACCAUGAGCGNN 32 1582-1600 UGGCAUCCUGGCUUGCCUCNN 2156 GAGGCAAGCCAGGAUGCCANN 32 1583-1601 GGCAUCCUGGCUUGCCUCCNN 2157 GGAGGCAAGCCAGGAUGCCNN 32 1584-1602 GCAUCCUGGCUUGCCUCCANN 2158 UGGAGGCAAGCCAGGAUGCNN 32 1585-1603 CAUCCUGGCUUGCCUCCAGNN 2159 CUGGAGGCAAGCCAGGAUGNN 32 1586-1604 AUCCUGGCUUGCCUCCAGUNN 2160 ACUGGAGGCAAGCCAGGAUNN 32 1587-1605 UCCUGGCUUGCCUCCAGUUNN 2161 AACUGGAGGCAAGCCAGGANN 32 1588-1606 CCUGGCUUGCCUCCAGUUUNN 2162 AAACUGGAGGCAAGCCAGGNN 32 1589-1607 CUGGCUUGCCUCCAGUUUUNN 2163 AAAACUGGAGGCAAGCCAGNN 32 1590-1608 UGGCUUGCCUCCAGUUUUANN 2164 UAAAACUGGAGGCAAGCCANN 32 1591-1609 GGCUUGCCUCCAGUUUUAGNN 2165 CUAAAACUGGAGGCAAGCCNN 32 159-177 GCUCAUGGUGCCAGCCCAGNN 2166 CUGGGCUGGCACCAUGAGCNN 32 1592-1610 GCUUGCCUCCAGUUUUAGGNN 2167 CCUAAAACUGGAGGCAAGCNN 32 1593-1611 CUUGCCUCCAGUUUUAGGUNN 2168 ACCUAAAACUGGAGGCAAGNN 32 1594-1612 UUGCCUCCAGUUUUAGGUCNN 2169 GACCUAAAACUGGAGGCAANN 32 1595-1613 UGCCUCCAGUUUUAGGUCCNN 2170 GGACCUAAAACUGGAGGCANN 32 160-178 CUCAUGGUGCCAGCCCAGANN 2171 UCUGGGCUGGCACCAUGAGNN 32 161-179 UCAUGGUGCCAGCCCAGAGNN 2172 CUCUGGGCUGGCACCAUGANN 32 1615-1633 UUAGUUUGCUUCUGUAAGCNN 2173 GCUUACAGAAGCAAACUAANN 32 1616-1634 UAGUUUGCUUCUGUAAGCANN 2174 UGCUUACAGAAGCAAACUANN 32 1617-1635 AGUUUGCUUCUGUAAGCAANN 2175 UUGCUUACAGAAGCAAACUNN 32 162-180 CAUGGUGCCAGCCCAGAGANN 2176 UCUCUGGGCUGGCACCAUGNN 32 163-181 AUGGUGCCAGCCCAGAGAGNN 2177 CUCUCUGGGCUGGCACCAUNN 32 1639-1657 GAACACCUGCUGAGGGGGCNN 2178 GCCCCCUCAGCAGGUGUUCNN 32 1640-1658 AACACCUGCUGAGGGGGCUNN 2179 AGCCCCCUCAGCAGGUGUUNN 32 1641-1659 ACACCUGCUGAGGGGGCUCNN 2180 GAGCCCCCUCAGCAGGUGUNN 32 164-182 UGGUGCCAGCCCAGAGAGGNN 2181 CCUCUCUGGGCUGGCACCANN 32 1642-1660 CACCUGCUGAGGGGGCUCUNN 2182 AGAGCCCCCUCAGCAGGUGNN 32 1643-1661 ACCUGCUGAGGGGGCUCUUNN 2183 AAGAGCCCCCUCAGCAGGUNN 32 1644-1662 CCUGCUGAGGGGGCUCUUUNN 2184 AAAGAGCCCCCUCAGCAGGNN 32 1645-1663 CUGCUGAGGGGGCUCUUUCNN 2185 GAAAGAGCCCCCUCAGCAGNN 32 1646-1664 UGCUGAGGGGGCUCUUUCCNN 2186 GGAAAGAGCCCCCUCAGCANN 32 1647-1665 GCUGAGGGGGCUCUUUCCCNN 2187 GGGAAAGAGCCCCCUCAGCNN 32 1648-1666 CUGAGGGGGCUCUUUCCCUNN 2188 AGGGAAAGAGCCCCCUCAGNN 32 1649-1667 UGAGGGGGCUCUUUCCCUCNN 2189 GAGGGAAAGAGCCCCCUCANN 32 1650-1668 GAGGGGGCUCUUUCCCUCANN 2190 UGAGGGAAAGAGCCCCCUCNN 32 165-183 GGUGCCAGCCCAGAGAGGGNN 2191 CCCUCUCUGGGCUGGCACCNN 32 166-184 GUGCCAGCCCAGAGAGGGGNN 2192 CCCCUCUCUGGGCUGGCACNN 32 1670-1688 GUAUACUUCAAGUAAGAUCNN 2193 GAUCUUACUUGAAGUAUACNN 32 1671-1689 UAUACUUCAAGUAAGAUCANN 2194 UGAUCUUACUUGAAGUAUANN 32 167-185 UGCCAGCCCAGAGAGGGGCNN 2195 GCCCCUCUCUGGGCUGGCANN 32 1672-1690 AUACUUCAAGUAAGAUCAANN 2196 UUGAUCUUACUUGAAGUAUNN 32 1673-1691 UACUUCAAGUAAGAUCAAGNN 2197 CUUGAUCUUACUUGAAGUANN 32 1674-1692 ACUUCAAGUAAGAUCAAGANN 2198 UCUUGAUCUUACUUGAAGUNN 32 1675-1693 CUUCAAGUAAGAUCAAGAANN 2199 UUCUUGAUCUUACUUGAAGNN 32 1676-1694 UUCAAGUAAGAUCAAGAAUNN 2200 AUUCUUGAUCUUACUUGAANN 32 1677-1695 UCAAGUAAGAUCAAGAAUCNN 2201 GAUUCUUGAUCUUACUUGANN 32 1678-1696 CAAGUAAGAUCAAGAAUCUNN 2202 AGAUUCUUGAUCUUACUUGNN 32 1679-1697 AAGUAAGAUCAAGAAUCUUNN 2203 AAGAUUCUUGAUCUUACUUNN 32 1680-1698 AGUAAGAUCAAGAAUCUUUNN 2204 AAAGAUUCUUGAUCUUACUNN 32 1681-1699 GUAAGAUCAAGAAUCUUUUNN 2205 AAAAGAUUCUUGAUCUUACNN 32 1682-1700 UAAGAUCAAGAAUCUUUUGNN 2206 CAAAAGAUUCUUGAUCUUANN 32 1683-1701 AAGAUCAAGAAUCUUUUGUNN 2207 ACAAAAGAUUCUUGAUCUUNN 32 1684-1702 AGAUCAAGAAUCUUUUGUGNN 2208 CACAAAAGAUUCUUGAUCUNN 32 1685-1703 GAUCAAGAAUCUUUUGUGANN 2209 UCACAAAAGAUUCUUGAUCNN 32 1686-1704 AUCAAGAAUCUUUUGUGAANN 2210 UUCACAAAAGAUUCUUGAUNN 32 1687-1705 UCAAGAAUCUUUUGUGAAANN 2211 UUUCACAAAAGAUUCUUGANN 32 1707-1725 UAUAGAAAUUUACUAUGUANN 2212 UACAUAGUAAAUUUCUAUANN 32 1708-1726 AUAGAAAUUUACUAUGUAANN 2213 UUACAUAGUAAAUUUCUAUNN 32 1709-1727 UAGAAAUUUACUAUGUAAANN 2214 UUUACAUAGUAAAUUUCUANN 32 1710-1728 AGAAAUUUACUAUGUAAAUNN 2215 AUUUACAUAGUAAAUUUCUNN 32 1711-1729 GAAAUUUACUAUGUAAAUGNN 2216 CAUUUACAUAGUAAAUUUCNN 32 1712-1730 AAAUUUACUAUGUAAAUGCNN 2217 GCAUUUACAUAGUAAAUUUNN 32 1713-1731 AAUUUACUAUGUAAAUGCUNN 2218 AGCAUUUACAUAGUAAAUUNN 32 1714-1732 AUUUACUAUGUAAAUGCUUNN 2219 AAGCAUUUACAUAGUAAAUNN 32 1715-1733 UUUACUAUGUAAAUGCUUGNN 2220 CAAGCAUUUACAUAGUAAANN 32 1716-1734 UUACUAUGUAAAUGCUUGANN 2221 UCAAGCAUUUACAUAGUAANN 32 1717-1735 UACUAUGUAAAUGCUUGAUNN 2222 AUCAAGCAUUUACAUAGUANN 32 1718-1736 ACUAUGUAAAUGCUUGAUGNN 2223 CAUCAAGCAUUUACAUAGUNN 32 1719-1737 CUAUGUAAAUGCUUGAUGGNN 2224 CCAUCAAGCAUUUACAUAGNN 32 1720-1738 UAUGUAAAUGCUUGAUGGANN 2225 UCCAUCAAGCAUUUACAUANN 32 1721-1739 AUGUAAAUGCUUGAUGGAANN 2226 UUCCAUCAAGCAUUUACAUNN 32 1722-1740 UGUAAAUGCUUGAUGGAAUNN 2227 AUUCCAUCAAGCAUUUACANN 32 1723-1741 GUAAAUGCUUGAUGGAAUUNN 2228 AAUUCCAUCAAGCAUUUACNN 32 1724-1742 UAAAUGCUUGAUGGAAUUUNN 2229 AAAUUCCAUCAAGCAUUUANN 32 1725-1743 AAAUGCUUGAUGGAAUUUUNN 2230 AAAAUUCCAUCAAGCAUUUNN 32 1726-1744 AAUGCUUGAUGGAAUUUUUNN 2231 AAAAAUUCCAUCAAGCAUUNN 32 1727-1745 AUGCUUGAUGGAAUUUUUUNN 2232 AAAAAAUUCCAUCAAGCAUNN 32 1728-1746 UGCUUGAUGGAAUUUUUUCNN 2233 GAAAAAAUUCCAUCAAGCANN 32 1729-1747 GCUUGAUGGAAUUUUUUCCNN 2234 GGAAAAAAUUCCAUCAAGCNN 32 1730-1748 CUUGAUGGAAUUUUUUCCUNN 2235 AGGAAAAAAUUCCAUCAAGNN 32 1731-1749 UUGAUGGAAUUUUUUCCUGNN 2236 CAGGAAAAAAUUCCAUCAANN 32 1732-1750 UGAUGGAAUUUUUUCCUGCNN 2237 GCAGGAAAAAAUUCCAUCANN 32 1733-1751 GAUGGAAUUUUUUCCUGCUNN 2238 AGCAGGAAAAAAUUCCAUCNN 32 1734-1752 AUGGAAUUUUUUCCUGCUANN 2239 UAGCAGGAAAAAAUUCCAUNN 32 1735-1753 UGGAAUUUUUUCCUGCUAGNN 2240 CUAGCAGGAAAAAAUUCCANN 32 1736-1754 GGAAUUUUUUCCUGCUAGUNN 2241 ACUAGCAGGAAAAAAUUCCNN 33 1737-1755 GAAUUUUUUCCUGCUAGUGNN 2242 CACUAGCAGGAAAAAAUUCNN 33 1738-1756 AAUUUUUUCCUGCUAGUGUNN 2243 ACACUAGCAGGAAAAAAUUNN 33 1739-1757 AUUUUUUCCUGCUAGUGUANN 2244 UACACUAGCAGGAAAAAAUNN 33 1740-1758 UUUUUUCCUGCUAGUGUAGNN 2245 CUACACUAGCAGGAAAAAANN 33 1741-1759 UUUUUCCUGCUAGUGUAGCNN 2246 GCUACACUAGCAGGAAAAANN 33 1742-1760 UUUUCCUGCUAGUGUAGCUNN 2247 AGCUACACUAGCAGGAAAANN 33 1743-1761 UUUCCUGCUAGUGUAGCUUNN 2248 AAGCUACACUAGCAGGAAANN 33 1744-1762 UUCCUGCUAGUGUAGCUUCNN 2249 GAAGCUACACUAGCAGGAANN 33 1745-1763 UCCUGCUAGUGUAGCUUCUNN 2250 AGAAGCUACACUAGCAGGANN 33 1746-1764 CCUGCUAGUGUAGCUUCUGNN 2251 CAGAAGCUACACUAGCAGGNN 33 1747-1765 CUGCUAGUGUAGCUUCUGANN 2252 UCAGAAGCUACACUAGCAGNN 33 1748-1766 UGCUAGUGUAGCUUCUGAANN 2253 UUCAGAAGCUACACUAGCANN 33 1749-1767 GCUAGUGUAGCUUCUGAAANN 2254 UUUCAGAAGCUACACUAGCNN 33 1750-1768 CUAGUGUAGCUUCUGAAAGNN 2255 CUUUCAGAAGCUACACUAGNN 33 1751-1769 UAGUGUAGCUUCUGAAAGGNN 2256 CCUUUCAGAAGCUACACUANN 33 1752-1770 AGUGUAGCUUCUGAAAGGUNN 2257 ACCUUUCAGAAGCUACACUNN 33 1753-1771 GUGUAGCUUCUGAAAGGUGNN 2258 CACCUUUCAGAAGCUACACNN 33 1754-1772 UGUAGCUUCUGAAAGGUGCNN 2259 GCACCUUUCAGAAGCUACANN 33 1755-1773 GUAGCUUCUGAAAGGUGCUNN 2260 AGCACCUUUCAGAAGCUACNN 33 1756-1774 UAGCUUCUGAAAGGUGCUUNN 2261 AAGCACCUUUCAGAAGCUANN 33 1757-1775 AGCUUCUGAAAGGUGCUUUNN 2262 AAAGCACCUUUCAGAAGCUNN 33 1758-1776 GCUUCUGAAAGGUGCUUUCNN 2263 GAAAGCACCUUUCAGAAGCNN 33 1777-1795 UCCAUUUAUUUAAAACUACNN 2264 GUAGUUUUAAAUAAAUGGANN 33 1778-1796 CCAUUUAUUUAAAACUACCNN 2265 GGUAGUUUUAAAUAAAUGGNN 33 1779-1797 CAUUUAUUUAAAACUACCCNN 2266 GGGUAGUUUUAAAUAAAUGNN 33 1780-1798 AUUUAUUUAAAACUACCCANN 2267 UGGGUAGUUUUAAAUAAAUNN 33 1781-1799 UUUAUUUAAAACUACCCAUNN 2268 AUGGGUAGUUUUAAAUAAANN 33 1782-1800 UUAUUUAAAACUACCCAUGNN 2269 CAUGGGUAGUUUUAAAUAANN 33 1783-1801 UAUUUAAAACUACCCAUGCNN 2270 GCAUGGGUAGUUUUAAAUANN 33 1784-1802 AUUUAAAACUACCCAUGCANN 2271 UGCAUGGGUAGUUUUAAAUNN 33 1785-1803 UUUAAAACUACCCAUGCAANN 2272 UUGCAUGGGUAGUUUUAAANN 33 1786-1804 UUAAAACUACCCAUGCAAUNN 2273 AUUGCAUGGGUAGUUUUAANN 33 1787-1805 UAAAACUACCCAUGCAAUUNN 2274 AAUUGCAUGGGUAGUUUUANN 33 1788-1806 AAAACUACCCAUGCAAUUANN 2275 UAAUUGCAUGGGUAGUUUUNN 33 1789-1807 AAACUACCCAUGCAAUUAANN 2276 UUAAUUGCAUGGGUAGUUUNN 33 1790-1808 AACUACCCAUGCAAUUAAANN 2277 UUUAAUUGCAUGGGUAGUUNN 33 1791-1809 ACUACCCAUGCAAUUAAAANN 2278 UUUUAAUUGCAUGGGUAGUNN 33 1792-1810 CUACCCAUGCAAUUAAAAGNN 2279 CUUUUAAUUGCAUGGGUAGNN 33 1793-1811 UACCCAUGCAAUUAAAAGGNN 2280 CCUUUUAAUUGCAUGGGUANN 33 1794-1812 ACCCAUGCAAUUAAAAGGUNN 2281 ACCUUUUAAUUGCAUGGGUNN 33 1795-1813 CCCAUGCAAUUAAAAGGUANN 2282 UACCUUUUAAUUGCAUGGGNN 33 1796-1814 CCAUGCAAUUAAAAGGUACNN 2283 GUACCUUUUAAUUGCAUGGNN 33 1797-1815 CAUGCAAUUAAAAGGUACANN 2284 UGUACCUUUUAAUUGCAUGNN 33 1798-1816 AUGCAAUUAAAAGGUACAANN 2285 UUGUACCUUUUAAUUGCAUNN 33 1799-1817 UGCAAUUAAAAGGUACAAUNN 2286 AUUGUACCUUUUAAUUGCANN 33 1800-1818 GCAAUUAAAAGGUACAAUGNN 2287 CAUUGUACCUUUUAAUUGCNN 33 1801-1819 CAAUUAAAAGGUACAAUGCNN 2288 GCAUUGUACCUUUUAAUUGNN 33 1802-1820 AAUUAAAAGGUACAAUGCANN 2289 UGCAUUGUACCUUUUAAUUNN 33 187-205 AGCCCGGAGGCAGCGAGCGNN 2290 CGCTCGCTGCCTCCGGGCTNN 33 188-206 GCCCGGAGGCAGCGAGCGGNN 2291 CCGCTCGCTGCCTCCGGGCNN 33 189-207 CCCGGAGGCAGCGAGCGGGNN 2292 CCCGCTCGCTGCCTCCGGGNN 33 190-208 CCGGAGGCAGCGAGCGGGGNN 2293 CCCCGCTCGCTGCCTCCGGNN 33 191-209 CGGAGGCAGCGAGCGGGGGNN 2294 CCCCCGCTCGCTGCCTCCGNN 33 192-210 GGAGGCAGCGAGCGGGGGGNN 2295 CCCCCCGCTCGCTGCCTCCNN 33 193-211 GAGGCAGCGAGCGGGGGGCNN 2296 GCCCCCCGCTCGCTGCCTCNN 33 194-212 AGGCAGCGAGCGGGGGGCUNN 2297 AGCCCCCCGCUCGCUGCCUNN 33 195-213 GGCAGCGAGCGGGGGGCUGNN 2298 CAGCCCCCCGCUCGCUGCCNN 33 196-214 GCAGCGAGCGGGGGGCUGCNN 2299 GCAGCCCCCCGCUCGCUGCNN 33 197-215 CAGCGAGCGGGGGGCUGCCNN 2300 GGCAGCCCCCCGCUCGCUGNN 33 198-216 AGCGAGCGGGGGGCUGCCCNN 2301 GGGCAGCCCCCCGCUCGCUNN 33 199-217 GCGAGCGGGGGGCUGCCCCNN 2302 GGGGCAGCCCCCCGCUCGCNN 33 200-218 CGAGCGGGGGGCUGCCCCANN 2303 UGGGGCAGCCCCCCGCUCGNN 33 201-219 GAGCGGGGGGCUGCCCCAGNN 2304 CUGGGGCAGCCCCCCGCUCNN 33 202-220 AGCGGGGGGCUGCCCCAGGNN 2305 CCUGGGGCAGCCCCCCGCUNN 33 203-221 GCGGGGGGCUGCCCCAGGCNN 2306 GCCUGGGGCAGCCCCCCGCNN 33 204-222 CGGGGGGCUGCCCCAGGCGNN 2307 CGCCUGGGGCAGCCCCCCGNN 33 205-223 GGGGGGCUGCCCCAGGCGCNN 2308 GCGCCUGGGGCAGCCCCCCNN 33 206-224 GGGGGCUGCCCCAGGCGCGNN 2309 CGCGCCUGGGGCAGCCCCCNN 33 207-225 GGGGCUGCCCCAGGCGCGCNN 2310 GCGCGCCUGGGGCAGCCCCNN 33 208-226 GGGCUGCCCCAGGCGCGCANN 2311 UGCGCGCCUGGGGCAGCCCNN 33 209-227 GGCUGCCCCAGGCGCGCAANN 2312 UUGCGCGCCUGGGGCAGCCNN 33 210-228 GCUGCCCCAGGCGCGCAAGNN 2313 CUUGCGCGCCUGGGGCAGCNN 33 211-229 CUGCCCCAGGCGCGCAAGCNN 2314 GCUUGCGCGCCUGGGGCAGNN 33 212-230 UGCCCCAGGCGCGCAAGCGNN 2315 CGCUUGCGCGCCUGGGGCANN 33 247-265 CUGAGCCCCGAGGAGAAGGNN 2316 CCUUCUCCUCGGGGCUCAGNN 33 248-266 UGAGCCCCGAGGAGAAGGCNN 2317 GCCUUCUCCUCGGGGCUCANN 33 249-267 GAGCCCCGAGGAGAAGGCGNN 2318 CGCCTTCTCCTCGGGGCTCNN 33 250-268 AGCCCCGAGGAGAAGGCGCNN 2319 GCGCCTTCTCCTCGGGGCTNN 33 251-269 GCCCCGAGGAGAAGGCGCUNN 2320 AGCGCCUUCUCCUCGGGGCNN 33 252-270 CCCCGAGGAGAAGGCGCUGNN 2321 CAGCGCCUUCUCCUCGGGGNN 33 253-271 CCCGAGGAGAAGGCGCUGANN 2322 UCAGCGCCUUCUCCUCGGGNN 33 254-272 CCGAGGAGAAGGCGCUGAGNN 2323 CUCAGCGCCUUCUCCUCGGNN 33 255-273 CGAGGAGAAGGCGCUGAGGNN 2324 CCUCAGCGCCUUCUCCUCGNN 33 256-274 GAGGAGAAGGCGCUGAGGANN 2325 UCCUCAGCGCCUUCUCCUCNN 33 257-275 AGGAGAAGGCGCUGAGGAGNN 2326 CUCCUCAGCGCCUUCUCCUNN 33 258-276 GGAGAAGGCGCUGAGGAGGNN 2327 CCUCCUCAGCGCCUUCUCCNN 33 259-277 GAGAAGGCGCUGAGGAGGANN 2328 UCCUCCUCAGCGCCUUCUCNN 33 260-278 AGAAGGCGCUGAGGAGGAANN 2329 UUCCUCCUCAGCGCCUUCUNN 33 261-279 GAAGGCGCUGAGGAGGAAANN 2330 UUUCCUCCUCAGCGCCUUCNN 33 262-280 AAGGCGCUGAGGAGGAAACNN 2331 GUUUCCUCCUCAGCGCCUUNN 33 263-281 AGGCGCUGAGGAGGAAACUNN 2332 AGUUUCCUCCUCAGCGCCUNN 33 264-282 GGCGCUGAGGAGGAAACUGNN 2333 CAGUUUCCUCCUCAGCGCCNN 33 265-283 GCGCUGAGGAGGAAACUGANN 2334 UCAGUUUCCUCCUCAGCGCNN 33 266-284 CGCUGAGGAGGAAACUGAANN 2335 UUCAGUUUCCUCCUCAGCGNN 33 267-285 GCUGAGGAGGAAACUGAAANN 2336 UUUCAGUUUCCUCCUCAGCNN 33 268-286 CUGAGGAGGAAACUGAAAANN 2337 UUUUCAGUUUCCUCCUCAGNN 33 269-287 UGAGGAGGAAACUGAAAAANN 2338 UUUUUCAGUUUCCUCCUCANN 33 270-288 GAGGAGGAAACUGAAAAACNN 2339 GUUUUUCAGUUUCCUCCUCNN 33 271-289 AGGAGGAAACUGAAAAACANN 2340 UGUUUUUCAGUUUCCUCCUNN 33 272-290 GGAGGAAACUGAAAAACAGNN 2341 CUGUUUUUCAGUUUCCUCCNN 34 273-291 GAGGAAACUGAAAAACAGANN 2342 UCUGUUUUUCAGUUUCCUCNN 34 274-292 AGGAAACUGAAAAACAGAGNN 2343 CUCUGUUUUUCAGUUUCCUNN 34 275-293 GGAAACUGAAAAACAGAGUNN 2344 ACUCUGUUUUUCAGUUUCCNN 34 276-294 GAAACUGAAAAACAGAGUANN 2345 UACUCUGUUUUUCAGUUUCNN 34 277-295 AAACUGAAAAACAGAGUAGNN 2346 CUACUCUGUUUUUCAGUUUNN 34 278-296 AACUGAAAAACAGAGUAGCNN 2347 GCUACUCUGUUUUUCAGUUNN 34 279-297 ACUGAAAAACAGAGUAGCANN 2348 UGCUACUCUGUUUUUCAGUNN 34 280-298 CUGAAAAACAGAGUAGCAGNN 2349 CUGCUACUCUGUUUUUCAGNN 34 281-299 UGAAAAACAGAGUAGCAGCNN 2350 GCUGCUACUCUGUUUUUCANN 34 282-300 GAAAAACAGAGUAGCAGCUNN 2351 AGCUGCUACUCUGUUUUUCNN 34 283-301 AAAAACAGAGUAGCAGCUCNN 2352 GAGCUGCUACUCUGUUUUUNN 34 284-302 AAAACAGAGUAGCAGCUCANN 2353 UGAGCUGCUACUCUGUUUUNN 34 285-303 AAACAGAGUAGCAGCUCAGNN 2354 CUGAGCUGCUACUCUGUUUNN 34 286-304 AACAGAGUAGCAGCUCAGANN 2355 UCUGAGCUGCUACUCUGUUNN 34 287-305 ACAGAGUAGCAGCUCAGACNN 2356 GUCUGAGCUGCUACUCUGUNN 34 288-306 CAGAGUAGCAGCUCAGACUNN 2357 AGUCUGAGCUGCUACUCUGNN 34 289-307 AGAGUAGCAGCUCAGACUGNN 2358 CAGUCUGAGCUGCUACUCUNN 34 290-308 GAGUAGCAGCUCAGACUGCNN 2359 GCAGUCUGAGCUGCUACUCNN 34 291-309 AGUAGCAGCUCAGACUGCCNN 2360 GGCAGUCUGAGCUGCUACUNN 34 292-310 GUAGCAGCUCAGACUGCCANN 2361 UGGCAGUCUGAGCUGCUACNN 34 293-311 UAGCAGCUCAGACUGCCAGNN 2362 CUGGCAGUCUGAGCUGCUANN 34 294-312 AGCAGCUCAGACUGCCAGANN 2363 UCUGGCAGUCUGAGCUGCUNN 34 295-313 GCAGCUCAGACUGCCAGAGNN 2364 CUCUGGCAGUCUGAGCUGCNN 34 296-314 CAGCUCAGACUGCCAGAGANN 2365 UCUCUGGCAGUCUGAGCUGNN 34 297-315 AGCUCAGACUGCCAGAGAUNN 2366 AUCUCUGGCAGUCUGAGCUNN 34 298-316 GCUCAGACUGCCAGAGAUCNN 2367 GAUCUCUGGCAGUCUGAGCNN 34 299-317 CUCAGACUGCCAGAGAUCGNN 2368 CGAUCUCUGGCAGUCUGAGNN 34 300-318 UCAGACUGCCAGAGAUCGANN 2369 UCGAUCUCUGGCAGUCUGANN 34 301-319 CAGACUGCCAGAGAUCGAANN 2370 UUCGAUCUCUGGCAGUCUGNN 34 302-320 AGACUGCCAGAGAUCGAAANN 2371 UUUCGAUCUCUGGCAGUCUNN 34 303-321 GACUGCCAGAGAUCGAAAGNN 2372 CUUUCGAUCUCUGGCAGUCNN 34 304-322 ACUGCCAGAGAUCGAAAGANN 2373 UCUUUCGAUCUCUGGCAGUNN 34 305-323 CUGCCAGAGAUCGAAAGAANN 2374 UUCUUUCGAUCUCUGGCAGNN 34 325-343 GCUCGAAUGAGUGAGCUGGNN 2375 CCAGCUCACUCAUUCGAGCNN 34 326-344 CUCGAAUGAGUGAGCUGGANN 2376 UCCAGCUCACUCAUUCGAGNN 34 327-345 UCGAAUGAGUGAGCUGGAANN 2377 UUCCAGCUCACUCAUUCGANN 34 328-346 CGAAUGAGUGAGCUGGAACNN 2378 GUUCCAGCUCACUCAUUCGNN 34 329-347 GAAUGAGUGAGCUGGAACANN 2379 UGUUCCAGCUCACUCAUUCNN 34 330-348 AAUGAGUGAGCUGGAACAGNN 2380 CUGUUCCAGCUCACUCAUUNN 34 331-349 AUGAGUGAGCUGGAACAGCNN 2381 GCUGUUCCAGCUCACUCAUNN 34 332-350 UGAGUGAGCUGGAACAGCANN 2382 UGCUGUUCCAGCUCACUCANN 34 333-351 GAGUGAGCUGGAACAGCAANN 2383 UUGCUGUUCCAGCUCACUCNN 34 334-352 AGUGAGCUGGAACAGCAAGNN 2384 CUUGCUGUUCCAGCUCACUNN 34 335-353 GUGAGCUGGAACAGCAAGUNN 2385 ACUUGCUGUUCCAGCUCACNN 34 336-354 UGAGCUGGAACAGCAAGUGNN 2386 CACUUGCUGUUCCAGCUCANN 34 337-355 GAGCUGGAACAGCAAGUGGNN 2387 CCACUUGCUGUUCCAGCUCNN 34 338-356 AGCUGGAACAGCAAGUGGUNN 2388 ACCACUUGCUGUUCCAGCUNN 34 339-357 GCUGGAACAGCAAGUGGUANN 2389 UACCACUUGCUGUUCCAGCNN 34 340-358 CUGGAACAGCAAGUGGUAGNN 2390 CUACCACUUGCUGUUCCAGNN 34 341-359 UGGAACAGCAAGUGGUAGANN 2391 UCUACCACUUGCUGUUCCANN 34 342-360 GGAACAGCAAGUGGUAGAUNN 2392 AUCUACCACUUGCUGUUCCNN 34 343-361 GAACAGCAAGUGGUAGAUUNN 2393 AAUCUACCACUUGCUGUUCNN 34 344-362 AACAGCAAGUGGUAGAUUUNN 2394 AAAUCUACCACUUGCUGUUNN 34 345-363 ACAGCAAGUGGUAGAUUUANN 2395 UAAAUCUACCACUUGCUGUNN 34 346-364 CAGCAAGUGGUAGAUUUAGNN 2396 CUAAAUCUACCACUUGCUGNN 34 347-365 AGCAAGUGGUAGAUUUAGANN 2397 UCUAAAUCUACCACUUGCUNN 34 348-366 GCAAGUGGUAGAUUUAGAANN 2398 UUCUAAAUCUACCACUUGCNN 34 349-367 CAAGUGGUAGAUUUAGAAGNN 2399 CUUCUAAAUCUACCACUUGNN 34 350-368 AAGUGGUAGAUUUAGAAGANN 2400 UCUUCUAAAUCUACCACUUNN 34 351-369 AGUGGUAGAUUUAGAAGAANN 2401 UUCUUCUAAAUCUACCACUNN 34 352-370 GUGGUAGAUUUAGAAGAAGNN 2402 CUUCUUCUAAAUCUACCACNN 34 353-371 UGGUAGAUUUAGAAGAAGANN 2403 UCUUCUUCUAAAUCUACCANN 34 354-372 GGUAGAUUUAGAAGAAGAGNN 2404 CUCUUCUUCUAAAUCUACCNN 34 355-373 GUAGAUUUAGAAGAAGAGANN 2405 UCUCUUCUUCUAAAUCUACNN 34 356-374 UAGAUUUAGAAGAAGAGAANN 2406 UUCUCUUCUUCUAAAUCUANN 34 357-375 AGAUUUAGAAGAAGAGAACNN 2407 GUUCUCUUCUUCUAAAUCUNN 34 358-376 GAUUUAGAAGAAGAGAACCNN 2408 GGUUCUCUUCUUCUAAAUCNN 34 359-377 AUUUAGAAGAAGAGAACCANN 2409 UGGUUCUCUUCUUCUAAAUNN 34 360-378 UUUAGAAGAAGAGAACCAANN 2410 UUGGUUCUCUUCUUCUAAANN 34 361-379 UUAGAAGAAGAGAACCAAANN 2411 UUUGGUUCUCUUCUUCUAANN 34 362-380 UAGAAGAAGAGAACCAAAANN 2412 UUUUGGUUCUCUUCUUCUANN 34 363-381 AGAAGAAGAGAACCAAAAANN 2413 TTTTTGGTTCTCTTCTTCTNN 34 364-382 GAAGAAGAGAACCAAAAACNN 2414 GTTTTTGGTTCTCTTCTTCNN 34 365-383 AAGAAGAGAACCAAAAACUNN 2415 AGUUUUUGGUUCUCUUCUUNN 34 366-384 AGAAGAGAACCAAAAACUUNN 2416 AAGUUUUUGGUUCUCUUCUNN 34 367-385 GAAGAGAACCAAAAACUUUNN 2417 AAAGUUUUUGGUUCUCUUCNN 34 368-386 AAGAGAACCAAAAACUUUUNN 2418 AAAAGUUUUUGGUUCUCUUNN 34 369-387 AGAGAACCAAAAACUUUUGNN 2419 CAAAAGUUUUUGGUUCUCUNN 34 370-388 GAGAACCAAAAACUUUUGCNN 2420 GCAAAAGUUUUUGGUUCUCNN 34 371-389 AGAACCAAAAACUUUUGCUNN 2421 AGCAAAAGUUUUUGGUUCUNN 34 372-390 GAACCAAAAACUUUUGCUANN 2422 UAGCAAAAGUUUUUGGUUCNN 34 373-391 AACCAAAAACUUUUGCUAGNN 2423 CUAGCAAAAGUUUUUGGUUNN 34 374-392 ACCAAAAACUUUUGCUAGANN 2424 UCUAGCAAAAGUUUUUGGUNN 34 375-393 CCAAAAACUUUUGCUAGAANN 2425 UUCUAGCAAAAGUUUUUGGNN 34 376-394 CAAAAACUUUUGCUAGAAANN 2426 UUUCUAGCAAAAGUUUUUGNN 34 377-395 AAAAACUUUUGCUAGAAAANN 2427 UUUUCUAGCAAAAGUUUUUNN 34 378-396 AAAACUUUUGCUAGAAAAUNN 2428 AUUUUCUAGCAAAAGUUUUNN 34 379-397 AAACUUUUGCUAGAAAAUCNN 2429 GAUUUUCUAGCAAAAGUUUNN 34 380-398 AACUUUUGCUAGAAAAUCANN 2430 UGAUUUUCUAGCAAAAGUUNN 34 381-399 ACUUUUGCUAGAAAAUCAGNN 2431 CUGAUUUUCUAGCAAAAGUNN 34 382-400 CUUUUGCUAGAAAAUCAGCNN 2432 GCUGAUUUUCUAGCAAAAGNN 34 383-401 UUUUGCUAGAAAAUCAGCUNN 2433 AGCUGAUUUUCUAGCAAAANN 34 384-402 UUUGCUAGAAAAUCAGCUUNN 2434 AAGCUGAUUUUCUAGCAAANN 34 385-403 UUGCUAGAAAAUCAGCUUUNN 2435 AAAGCUGAUUUUCUAGCAANN 34 386-404 UGCUAGAAAAUCAGCUUUUNN 2436 AAAAGCUGAUUUUCUAGCANN 34 387-405 GCUAGAAAAUCAGCUUUUANN 2437 UAAAAGCUGAUUUUCUAGCNN 34 388-406 CUAGAAAAUCAGCUUUUACNN 2438 GUAAAAGCUGAUUUUCUAGNN 34 389-407 UAGAAAAUCAGCUUUUACGNN 2439 CGUAAAAGCUGAUUUUCUANN 34 390-408 AGAAAAUCAGCUUUUACGANN 2440 UCGUAAAAGCUGAUUUUCUNN 34 391-409 GAAAAUCAGCUUUUACGAGNN 2441 CUCGUAAAAGCUGAUUUUCNN 35 392-410 AAAAUCAGCUUUUACGAGANN 2442 UCUCGUAAAAGCUGAUUUUNN 35 393-411 AAAUCAGCUUUUACGAGAGNN 2443 CUCUCGUAAAAGCUGAUUUNN 35 394-412 AAUCAGCUUUUACGAGAGANN 2444 UCUCUCGUAAAAGCUGAUUNN 35 395-413 AUCAGCUUUUACGAGAGAANN 2445 UUCUCUCGUAAAAGCUGAUNN 35 396-414 UCAGCUUUUACGAGAGAAANN 2446 UUUCUCUCGUAAAAGCUGANN 35 397-415 CAGCUUUUACGAGAGAAAANN 2447 UUUUCUCUCGUAAAAGCUGNN 35 398-416 AGCUUUUACGAGAGAAAACNN 2448 GUUUUCUCUCGUAAAAGCUNN 35 399-417 GCUUUUACGAGAGAAAACUNN 2449 AGUUUUCUCUCGUAAAAGCNN 35 400-418 CUUUUACGAGAGAAAACUCNN 2450 GAGUUUUCUCUCGUAAAAGNN 35 401-419 UUUUACGAGAGAAAACUCANN 2451 UGAGUUUUCUCUCGUAAAANN 35 421-439 GGCCUUGUAGUUGAGAACCNN 2452 GGUUCUCAACUACAAGGCCNN 35 422-440 GCCUUGUAGUUGAGAACCANN 2453 UGGUUCUCAACUACAAGGCNN 35 423-441 CCUUGUAGUUGAGAACCAGNN 2454 CUGGUUCUCAACUACAAGGNN 35 424-442 CUUGUAGUUGAGAACCAGGNN 2455 CCUGGUUCUCAACUACAAGNN 35 425-443 UUGUAGUUGAGAACCAGGANN 2456 UCCUGGUUCUCAACUACAANN 35 426-444 UGUAGUUGAGAACCAGGAGNN 2457 CUCCUGGUUCUCAACUACANN 35 427-445 GUAGUUGAGAACCAGGAGUNN 2458 ACUCCUGGUUCUCAACUACNN 35 428-446 UAGUUGAGAACCAGGAGUUNN 2459 AACUCCUGGUUCUCAACUANN 35 429-447 AGUUGAGAACCAGGAGUUANN 2460 UAACUCCUGGUUCUCAACUNN 35 430-448 GUUGAGAACCAGGAGUUAANN 2461 UUAACUCCUGGUUCUCAACNN 35 431-449 UUGAGAACCAGGAGUUAAGNN 2462 CUUAACUCCUGGUUCUCAANN 35 432-450 UGAGAACCAGGAGUUAAGANN 2463 UCUUAACUCCUGGUUCUCANN 35 433-451 GAGAACCAGGAGUUAAGACNN 2464 GUCUUAACUCCUGGUUCUCNN 35 434-452 AGAACCAGGAGUUAAGACANN 2465 UGUCUUAACUCCUGGUUCUNN 35 435-453 GAACCAGGAGUUAAGACAGNN 2466 CUGUCUUAACUCCUGGUUCNN 35 436-454 AACCAGGAGUUAAGACAGCNN 2467 GCUGUCUUAACUCCUGGUUNN 35 437-455 ACCAGGAGUUAAGACAGCGNN 2468 CGCUGUCUUAACUCCUGGUNN 35 438-456 CCAGGAGUUAAGACAGCGCNN 2469 GCGCUGUCUUAACUCCUGGNN 35 44-62 GAGCUAUGGUGGUGGUGGCNN 2470 GCCACCACCACCAUAGCUCNN 35 45-63 AGCUAUGGUGGUGGUGGCANN 2471 UGCCACCACCACCAUAGCUNN 35 458-476 UGGGGAUGGAUGCCCUGGUNN 2472 ACCAGGGCAUCCAUCCCCANN 35 459-477 GGGGAUGGAUGCCCUGGUUNN 2473 AACCAGGGCAUCCAUCCCCNN 35 460-478 GGGAUGGAUGCCCUGGUUGNN 2474 CAACCAGGGCAUCCAUCCCNN 35 461-479 GGAUGGAUGCCCUGGUUGCNN 2475 GCAACCAGGGCAUCCAUCCNN 35 462-480 GAUGGAUGCCCUGGUUGCUNN 2476 AGCAACCAGGGCAUCCAUCNN 35 46-64 GCUAUGGUGGUGGUGGCAGNN 2477 CUGCCACCACCACCAUAGCNN 35 47-65 CUAUGGUGGUGGUGGCAGCNN 2478 GCUGCCACCACCACCAUAGNN 35 482-500 AAGAGGAGGCGGAAGCCAANN 2479 TTGGCTTCCGCCTCCTCTTNN 35 483-501 AGAGGAGGCGGAAGCCAAGNN 2480 CTTGGCTTCCGCCTCCTCTNN 35 484-502 GAGGAGGCGGAAGCCAAGGNN 2481 CCTTGGCTTCCGCCTCCTCNN 35 485-503 AGGAGGCGGAAGCCAAGGGNN 2482 CCCTTGGCTTCCGCCTCCTNN 35 486-504 GGAGGCGGAAGCCAAGGGGNN 2483 CCCCTTGGCTTCCGCCTCCNN 35 48-66 UAUGGUGGUGGUGGCAGCCNN 2484 GGCUGCCACCACCACCAUANN 35 487-505 GAGGCGGAAGCCAAGGGGANN 2485 TCCCCTTGGCTTCCGCCTCNN 35 488-506 AGGCGGAAGCCAAGGGGAANN 2486 TTCCCCTTGGCTTCCGCCTNN 35 489-507 GGCGGAAGCCAAGGGGAAUNN 2487 AUUCCCCUUGGCUUCCGCCNN 35 490-508 GCGGAAGCCAAGGGGAAUGNN 2488 CAUUCCCCUUGGCUUCCGCNN 35 49-67 AUGGUGGUGGUGGCAGCCGNN 2489 CGGCUGCCACCACCACCAUNN 35 50-68 UGGUGGUGGUGGCAGCCGCNN 2490 GCGGCUGCCACCACCACCANN 35 510-528 AGUGAGGCCAGUGGCCGGGNN 2491 CCCGGCCACUGGCCUCACUNN 35 511-529 GUGAGGCCAGUGGCCGGGUNN 2492 ACCCGGCCACUGGCCUCACNN 35 512-530 UGAGGCCAGUGGCCGGGUCNN 2493 GACCCGGCCACUGGCCUCANN 35 513-531 GAGGCCAGUGGCCGGGUCUNN 2494 AGACCCGGCCACUGGCCUCNN 35 514-532 AGGCCAGUGGCCGGGUCUGNN 2495 CAGACCCGGCCACUGGCCUNN 35 515-533 GGCCAGUGGCCGGGUCUGCNN 2496 GCAGACCCGGCCACUGGCCNN 35 516-534 GCCAGUGGCCGGGUCUGCUNN 2497 AGCAGACCCGGCCACUGGCNN 35 517-535 CCAGUGGCCGGGUCUGCUGNN 2498 CAGCAGACCCGGCCACUGGNN 35 518-536 CAGUGGCCGGGUCUGCUGANN 2499 UCAGCAGACCCGGCCACUGNN 35 519-537 AGUGGCCGGGUCUGCUGAGNN 2500 CUCAGCAGACCCGGCCACUNN 35 520-538 GUGGCCGGGUCUGCUGAGUNN 2501 ACUCAGCAGACCCGGCCACNN 35 521-539 UGGCCGGGUCUGCUGAGUCNN 2502 GACUCAGCAGACCCGGCCANN 35 522-540 GGCCGGGUCUGCUGAGUCCNN 2503 GGACUCAGCAGACCCGGCCNN 35 523-541 GCCGGGUCUGCUGAGUCCGNN 2504 CGGACUCAGCAGACCCGGCNN 35 524-542 CCGGGUCUGCUGAGUCCGCNN 2505 GCGGACUCAGCAGACCCGGNN 35 525-543 CGGGUCUGCUGAGUCCGCANN 2506 UGCGGACUCAGCAGACCCGNN 35 526-544 GGGUCUGCUGAGUCCGCAGNN 2507 CUGCGGACUCAGCAGACCCNN 35 574-592 GUGCAGGCCCAGUUGUCACNN 2508 GUGACAACUGGGCCUGCACNN 35 575-593 UGCAGGCCCAGUUGUCACCNN 2509 GGUGACAACUGGGCCUGCANN 35 576-594 GCAGGCCCAGUUGUCACCCNN 2510 GGGUGACAACUGGGCCUGCNN 35 577-595 CAGGCCCAGUUGUCACCCCNN 2511 GGGGUGACAACUGGGCCUGNN 35 578-596 AGGCCCAGUUGUCACCCCUNN 2512 AGGGGUGACAACUGGGCCUNN 35 579-597 GGCCCAGUUGUCACCCCUCNN 2513 GAGGGGUGACAACUGGGCCNN 35 580-598 GCCCAGUUGUCACCCCUCCNN 2514 GGAGGGGUGACAACUGGGCNN 35 581-599 CCCAGUUGUCACCCCUCCANN 2515 UGGAGGGGUGACAACUGGGNN 35 582-600 CCAGUUGUCACCCCUCCAGNN 2516 CUGGAGGGGUGACAACUGGNN 35 583-601 CAGUUGUCACCCCUCCAGANN 2517 UCUGGAGGGGUGACAACUGNN 35 584-602 AGUUGUCACCCCUCCAGAANN 2518 UUCUGGAGGGGUGACAACUNN 35 585-603 GUUGUCACCCCUCCAGAACNN 2519 GUUCUGGAGGGGUGACAACNN 35 586-604 UUGUCACCCCUCCAGAACANN 2520 UGUUCUGGAGGGGUGACAANN 35 587-605 UGUCACCCCUCCAGAACAUNN 2521 AUGUUCUGGAGGGGUGACANN 35 588-606 GUCACCCCUCCAGAACAUCNN 2522 GAUGUUCUGGAGGGGUGACNN 35 589-607 UCACCCCUCCAGAACAUCUNN 2523 AGAUGUUCUGGAGGGGUGANN 35 590-608 CACCCCUCCAGAACAUCUCNN 2524 GAGAUGUUCUGGAGGGGUGNN 35 591-609 ACCCCUCCAGAACAUCUCCNN 2525 GGAGAUGUUCUGGAGGGGUNN 35 592-610 CCCCUCCAGAACAUCUCCCNN 2526 GGGAGAUGUUCUGGAGGGGNN 35 593-611 CCCUCCAGAACAUCUCCCCNN 2527 GGGGAGAUGUUCUGGAGGGNN 35 594-612 CCUCCAGAACAUCUCCCCANN 2528 UGGGGAGAUGUUCUGGAGGNN 35 595-613 CUCCAGAACAUCUCCCCAUNN 2529 AUGGGGAGAUGUUCUGGAGNN 35 596-614 UCCAGAACAUCUCCCCAUGNN 2530 CAUGGGGAGAUGUUCUGGANN 35 597-615 CCAGAACAUCUCCCCAUGGNN 2531 CCAUGGGGAGAUGUUCUGGNN 35 598-616 CAGAACAUCUCCCCAUGGANN 2532 UCCAUGGGGAGAUGUUCUGNN 35 599-617 AGAACAUCUCCCCAUGGAUNN 2533 AUCCAUGGGGAGAUGUUCUNN 35 600-618 GAACAUCUCCCCAUGGAUUNN 2534 AAUCCAUGGGGAGAUGUUCNN 35 601-619 AACAUCUCCCCAUGGAUUCNN 2535 GAAUCCAUGGGGAGAUGUUNN 35 602-620 ACAUCUCCCCAUGGAUUCUNN 2536 AGAAUCCAUGGGGAGAUGUNN 35 603-621 CAUCUCCCCAUGGAUUCUGNN 2537 CAGAAUCCAUGGGGAGAUGNN 35 604-622 AUCUCCCCAUGGAUUCUGGNN 2538 CCAGAAUCCAUGGGGAGAUNN 35 605-623 UCUCCCCAUGGAUUCUGGCNN 2539 GCCAGAAUCCAUGGGGAGANN 35 606-624 CUCCCCAUGGAUUCUGGCGNN 2540 CGCCAGAAUCCAUGGGGAGNN 35 607-625 UCCCCAUGGAUUCUGGCGGNN 2541 CCGCCAGAAUCCAUGGGGANN 36 608-626 CCCCAUGGAUUCUGGCGGUNN 2542 ACCGCCAGAAUCCAUGGGGNN 36 609-627 CCCAUGGAUUCUGGCGGUANN 2543 UACCGCCAGAAUCCAUGGGNN 36 610-628 CCAUGGAUUCUGGCGGUAUNN 2544 AUACCGCCAGAAUCCAUGGNN 36 611-629 CAUGGAUUCUGGCGGUAUUNN 2545 AAUACCGCCAGAAUCCAUGNN 36 612-630 AUGGAUUCUGGCGGUAUUGNN 2546 CAAUACCGCCAGAAUCCAUNN 36 613-631 UGGAUUCUGGCGGUAUUGANN 2547 UCAAUACCGCCAGAAUCCANN 36 614-632 GGAUUCUGGCGGUAUUGACNN 2548 GUCAAUACCGCCAGAAUCCNN 36 615-633 GAUUCUGGCGGUAUUGACUNN 2549 AGUCAAUACCGCCAGAAUCNN 36 616-634 AUUCUGGCGGUAUUGACUCNN 2550 GAGUCAAUACCGCCAGAAUNN 36 617-635 UUCUGGCGGUAUUGACUCUNN 2551 AGAGUCAAUACCGCCAGAANN 36 618-636 UCUGGCGGUAUUGACUCUUNN 2552 AAGAGUCAAUACCGCCAGANN 36 619-637 CUGGCGGUAUUGACUCUUCNN 2553 GAAGAGUCAAUACCGCCAGNN 36 620-638 UGGCGGUAUUGACUCUUCANN 2554 UGAAGAGUCAAUACCGCCANN 36 621-639 GGCGGUAUUGACUCUUCAGNN 2555 CUGAAGAGUCAAUACCGCCNN 36 622-640 GCGGUAUUGACUCUUCAGANN 2556 UCUGAAGAGUCAAUACCGCNN 36 623-641 CGGUAUUGACUCUUCAGAUNN 2557 AUCUGAAGAGUCAAUACCGNN 36 624-642 GGUAUUGACUCUUCAGAUUNN 2558 AAUCUGAAGAGUCAAUACCNN 36 625-643 GUAUUGACUCUUCAGAUUCNN 2559 GAAUCUGAAGAGUCAAUACNN 36 626-644 UAUUGACUCUUCAGAUUCANN 2560 UGAAUCUGAAGAGUCAAUANN 36 627-645 AUUGACUCUUCAGAUUCAGNN 2561 CUGAAUCUGAAGAGUCAAUNN 36 628-646 UUGACUCUUCAGAUUCAGANN 2562 UCUGAAUCUGAAGAGUCAANN 36 629-647 UGACUCUUCAGAUUCAGAGNN 2563 CUCUGAAUCUGAAGAGUCANN 36 630-648 GACUCUUCAGAUUCAGAGUNN 2564 ACUCUGAAUCUGAAGAGUCNN 36 631-649 ACUCUUCAGAUUCAGAGUCNN 2565 GACUCUGAAUCUGAAGAGUNN 36 632-650 CUCUUCAGAUUCAGAGUCUNN 2566 AGACUCUGAAUCUGAAGAGNN 36 633-651 UCUUCAGAUUCAGAGUCUGNN 2567 CAGACUCUGAAUCUGAAGANN 36 634-652 CUUCAGAUUCAGAGUCUGANN 2568 UCAGACUCUGAAUCUGAAGNN 36 635-653 UUCAGAUUCAGAGUCUGAUNN 2569 AUCAGACUCUGAAUCUGAANN 36 636-654 UCAGAUUCAGAGUCUGAUANN 2570 UAUCAGACUCUGAAUCUGANN 36 637-655 CAGAUUCAGAGUCUGAUAUNN 2571 AUAUCAGACUCUGAAUCUGNN 36 638-656 AGAUUCAGAGUCUGAUAUCNN 2572 GAUAUCAGACUCUGAAUCUNN 36 639-657 GAUUCAGAGUCUGAUAUCCNN 2573 GGAUAUCAGACUCUGAAUCNN 36 640-658 AUUCAGAGUCUGAUAUCCUNN 2574 AGGAUAUCAGACUCUGAAUNN 36 641-659 UUCAGAGUCUGAUAUCCUGNN 2575 CAGGAUAUCAGACUCUGAANN 36 642-660 UCAGAGUCUGAUAUCCUGUNN 2576 ACAGGAUAUCAGACUCUGANN 36 643-661 CAGAGUCUGAUAUCCUGUUNN 2577 AACAGGAUAUCAGACUCUGNN 36 644-662 AGAGUCUGAUAUCCUGUUGNN 2578 CAACAGGAUAUCAGACUCUNN 36 645-663 GAGUCUGAUAUCCUGUUGGNN 2579 CCAACAGGAUAUCAGACUCNN 36 646-664 AGUCUGAUAUCCUGUUGGGNN 2580 CCCAACAGGAUAUCAGACUNN 36 647-665 GUCUGAUAUCCUGUUGGGCNN 2581 GCCCAACAGGAUAUCAGACNN 36 648-666 UCUGAUAUCCUGUUGGGCANN 2582 UGCCCAACAGGAUAUCAGANN 36 649-667 CUGAUAUCCUGUUGGGCAUNN 2583 AUGCCCAACAGGAUAUCAGNN 36 650-668 UGAUAUCCUGUUGGGCAUUNN 2584 AAUGCCCAACAGGAUAUCANN 36 651-669 GAUAUCCUGUUGGGCAUUCNN 2585 GAAUGCCCAACAGGAUAUCNN 36 652-670 AUAUCCUGUUGGGCAUUCUNN 2586 AGAAUGCCCAACAGGAUAUNN 36 653-671 UAUCCUGUUGGGCAUUCUGNN 2587 CAGAAUGCCCAACAGGAUANN 36 654-672 AUCCUGUUGGGCAUUCUGGNN 2588 CCAGAAUGCCCAACAGGAUNN 36 655-673 UCCUGUUGGGCAUUCUGGANN 2589 UCCAGAAUGCCCAACAGGANN 36 656-674 CCUGUUGGGCAUUCUGGACNN 2590 GUCCAGAAUGCCCAACAGGNN 36 657-675 CUGUUGGGCAUUCUGGACANN 2591 UGUCCAGAAUGCCCAACAGNN 36 658-676 UGUUGGGCAUUCUGGACAANN 2592 UUGUCCAGAAUGCCCAACANN 36 659-677 GUUGGGCAUUCUGGACAACNN 2593 GUUGUCCAGAAUGCCCAACNN 36 660-678 UUGGGCAUUCUGGACAACUNN 2594 AGUUGUCCAGAAUGCCCAANN 36 661-679 UGGGCAUUCUGGACAACUUNN 2595 AAGUUGUCCAGAAUGCCCANN 36 662-680 GGGCAUUCUGGACAACUUGNN 2596 CAAGUUGUCCAGAAUGCCCNN 36 663-681 GGCAUUCUGGACAACUUGGNN 2597 CCAAGUUGUCCAGAAUGCCNN 36 664-682 GCAUUCUGGACAACUUGGANN 2598 UCCAAGUUGUCCAGAAUGCNN 36 665-683 CAUUCUGGACAACUUGGACNN 2599 GUCCAAGUUGUCCAGAAUGNN 36 666-684 AUUCUGGACAACUUGGACCNN 2600 GGUCCAAGUUGUCCAGAAUNN 36 667-685 UUCUGGACAACUUGGACCCNN 2601 GGGUCCAAGUUGUCCAGAANN 36 668-686 UCUGGACAACUUGGACCCANN 2602 UGGGUCCAAGUUGUCCAGANN 36 669-687 CUGGACAACUUGGACCCAGNN 2603 CUGGGUCCAAGUUGUCCAGNN 36 670-688 UGGACAACUUGGACCCAGUNN 2604 ACUGGGUCCAAGUUGUCCANN 36 671-689 GGACAACUUGGACCCAGUCNN 2605 GACUGGGUCCAAGUUGUCCNN 36 672-690 GACAACUUGGACCCAGUCANN 2606 UGACUGGGUCCAAGUUGUCNN 36 673-691 ACAACUUGGACCCAGUCAUNN 2607 AUGACUGGGUCCAAGUUGUNN 36 674-692 CAACUUGGACCCAGUCAUGNN 2608 CAUGACUGGGUCCAAGUUGNN 36 675-693 AACUUGGACCCAGUCAUGUNN 2609 ACAUGACUGGGUCCAAGUUNN 36 676-694 ACUUGGACCCAGUCAUGUUNN 2610 AACAUGACUGGGUCCAAGUNN 36 677-695 CUUGGACCCAGUCAUGUUCNN 2611 GAACAUGACUGGGUCCAAGNN 36 678-696 UUGGACCCAGUCAUGUUCUNN 2612 AGAACAUGACUGGGUCCAANN 36 679-697 UGGACCCAGUCAUGUUCUUNN 2613 AAGAACAUGACUGGGUCCANN 36 680-698 GGACCCAGUCAUGUUCUUCNN 2614 GAAGAACAUGACUGGGUCCNN 36 681-699 GACCCAGUCAUGUUCUUCANN 2615 UGAAGAACAUGACUGGGUCNN 36 682-700 ACCCAGUCAUGUUCUUCAANN 2616 UUGAAGAACAUGACUGGGUNN 36 683-701 CCCAGUCAUGUUCUUCAAANN 2617 UUUGAAGAACAUGACUGGGNN 36 684-702 CCAGUCAUGUUCUUCAAAUNN 2618 AUUUGAAGAACAUGACUGGNN 36 685-703 CAGUCAUGUUCUUCAAAUGNN 2619 CAUUUGAAGAACAUGACUGNN 36 686-704 AGUCAUGUUCUUCAAAUGCNN 2620 GCAUUUGAAGAACAUGACUNN 36 687-705 GUCAUGUUCUUCAAAUGCCNN 2621 GGCAUUUGAAGAACAUGACNN 36 688-706 UCAUGUUCUUCAAAUGCCCNN 2622 GGGCAUUUGAAGAACAUGANN 36 689-707 CAUGUUCUUCAAAUGCCCUNN 2623 AGGGCAUUUGAAGAACAUGNN 36 690-708 AUGUUCUUCAAAUGCCCUUNN 2624 AAGGGCAUUUGAAGAACAUNN 36 691-709 UGUUCUUCAAAUGCCCUUCNN 2625 GAAGGGCAUUUGAAGAACANN 36 692-710 GUUCUUCAAAUGCCCUUCCNN 2626 GGAAGGGCAUUUGAAGAACNN 36 693-711 UUCUUCAAAUGCCCUUCCCNN 2627 GGGAAGGGCAUUUGAAGAANN 36 694-712 UCUUCAAAUGCCCUUCCCCNN 2628 GGGGAAGGGCAUUUGAAGANN 36 695-713 CUUCAAAUGCCCUUCCCCANN 2629 UGGGGAAGGGCAUUUGAAGNN 36 696-714 UUCAAAUGCCCUUCCCCAGNN 2630 CUGGGGAAGGGCAUUUGAANN 36 697-715 UCAAAUGCCCUUCCCCAGANN 2631 UCUGGGGAAGGGCAUUUGANN 36 698-716 CAAAUGCCCUUCCCCAGAGNN 2632 CUCUGGGGAAGGGCAUUUGNN 36 718-736 CUGCCAGCCUGGAGGAGCUNN 2633 AGCUCCUCCAGGCUGGCAGNN 36 719-737 UGCCAGCCUGGAGGAGCUCNN 2634 GAGCUCCUCCAGGCUGGCANN 36 720-738 GCCAGCCUGGAGGAGCUCCNN 2635 GGAGCUCCUCCAGGCUGGCNN 36 721-739 CCAGCCUGGAGGAGCUCCCNN 2636 GGGAGCUCCUCCAGGCUGGNN 36 722-740 CAGCCUGGAGGAGCUCCCANN 2637 UGGGAGCUCCUCCAGGCUGNN 36 723-741 AGCCUGGAGGAGCUCCCAGNN 2638 CUGGGAGCUCCUCCAGGCUNN 36 724-742 GCCUGGAGGAGCUCCCAGANN 2639 UCUGGGAGCUCCUCCAGGCNN 36 725-743 CCUGGAGGAGCUCCCAGAGNN 2640 CUCUGGGAGCUCCUCCAGGNN 36 726-744 CUGGAGGAGCUCCCAGAGGNN 2641 CCUCUGGGAGCUCCUCCAGNN 37 727-745 UGGAGGAGCUCCCAGAGGUNN 2642 ACCUCUGGGAGCUCCUCCANN 37 728-746 GGAGGAGCUCCCAGAGGUCNN 2643 GACCUCUGGGAGCUCCUCCNN 37 729-747 GAGGAGCUCCCAGAGGUCUNN 2644 AGACCUCUGGGAGCUCCUCNN 37 730-748 AGGAGCUCCCAGAGGUCUANN 2645 UAGACCUCUGGGAGCUCCUNN 37 731-749 GGAGCUCCCAGAGGUCUACNN 2646 GUAGACCUCUGGGAGCUCCNN 37 732-750 GAGCUCCCAGAGGUCUACCNN 2647 GGUAGACCUCUGGGAGCUCNN 37 733-751 AGCUCCCAGAGGUCUACCCNN 2648 GGGUAGACCUCUGGGAGCUNN 37 734-752 GCUCCCAGAGGUCUACCCANN 2649 UGGGUAGACCUCUGGGAGCNN 37 735-753 CUCCCAGAGGUCUACCCAGNN 2650 CUGGGUAGACCUCUGGGAGNN 37 736-754 UCCCAGAGGUCUACCCAGANN 2651 UCUGGGUAGACCUCUGGGANN 37 737-755 CCCAGAGGUCUACCCAGAANN 2652 UUCUGGGUAGACCUCUGGGNN 37 738-756 CCAGAGGUCUACCCAGAAGNN 2653 CUUCUGGGUAGACCUCUGGNN 37 739-757 CAGAGGUCUACCCAGAAGGNN 2654 CCUUCUGGGUAGACCUCUGNN 37 740-758 AGAGGUCUACCCAGAAGGANN 2655 UCCUUCUGGGUAGACCUCUNN 37 741-759 GAGGUCUACCCAGAAGGACNN 2656 GUCCUUCUGGGUAGACCUCNN 37 742-760 AGGUCUACCCAGAAGGACCNN 2657 GGUCCUUCUGGGUAGACCUNN 37 743-761 GGUCUACCCAGAAGGACCCNN 2658 GGGUCCUUCUGGGUAGACCNN 37 744-762 GUCUACCCAGAAGGACCCANN 2659 UGGGUCCUUCUGGGUAGACNN 37 745-763 UCUACCCAGAAGGACCCAGNN 2660 CUGGGUCCUUCUGGGUAGANN 37 746-764 CUACCCAGAAGGACCCAGUNN 2661 ACUGGGUCCUUCUGGGUAGNN 37 747-765 UACCCAGAAGGACCCAGUUNN 2662 AACUGGGUCCUUCUGGGUANN 37 748-766 ACCCAGAAGGACCCAGUUCNN 2663 GAACUGGGUCCUUCUGGGUNN 37 749-767 CCCAGAAGGACCCAGUUCCNN 2664 GGAACUGGGUCCUUCUGGGNN 37 750-768 CCAGAAGGACCCAGUUCCUNN 2665 AGGAACUGGGUCCUUCUGGNN 37 751-769 CAGAAGGACCCAGUUCCUUNN 2666 AAGGAACUGGGUCCUUCUGNN 37 752-770 AGAAGGACCCAGUUCCUUANN 2667 UAAGGAACUGGGUCCUUCUNN 37 753-771 GAAGGACCCAGUUCCUUACNN 2668 GUAAGGAACUGGGUCCUUCNN 37 754-772 AAGGACCCAGUUCCUUACCNN 2669 GGUAAGGAACUGGGUCCUUNN 37 755-773 AGGACCCAGUUCCUUACCANN 2670 UGGUAAGGAACUGGGUCCUNN 37 756-774 GGACCCAGUUCCUUACCAGNN 2671 CUGGUAAGGAACUGGGUCCNN 37 757-775 GACCCAGUUCCUUACCAGCNN 2672 GCUGGUAAGGAACUGGGUCNN 37 758-776 ACCCAGUUCCUUACCAGCCNN 2673 GGCUGGUAAGGAACUGGGUNN 37 759-777 CCCAGUUCCUUACCAGCCUNN 2674 AGGCUGGUAAGGAACUGGGNN 37 760-778 CCAGUUCCUUACCAGCCUCNN 2675 GAGGCUGGUAAGGAACUGGNN 37 761-779 CAGUUCCUUACCAGCCUCCNN 2676 GGAGGCUGGUAAGGAACUGNN 37 762-780 AGUUCCUUACCAGCCUCCCNN 2677 GGGAGGCUGGUAAGGAACUNN 37 763-781 GUUCCUUACCAGCCUCCCUNN 2678 AGGGAGGCUGGUAAGGAACNN 37 764-782 UUCCUUACCAGCCUCCCUUNN 2679 AAGGGAGGCUGGUAAGGAANN 37 765-783 UCCUUACCAGCCUCCCUUUNN 2680 AAAGGGAGGCUGGUAAGGANN 37 766-784 CCUUACCAGCCUCCCUUUCNN 2681 GAAAGGGAGGCUGGUAAGGNN 37 767-785 CUUACCAGCCUCCCUUUCUNN 2682 AGAAAGGGAGGCUGGUAAGNN 37 768-786 UUACCAGCCUCCCUUUCUCNN 2683 GAGAAAGGGAGGCUGGUAANN 37 769-787 UACCAGCCUCCCUUUCUCUNN 2684 AGAGAAAGGGAGGCUGGUANN 37 770-788 ACCAGCCUCCCUUUCUCUGNN 2685 CAGAGAAAGGGAGGCUGGUNN 37 771-789 CCAGCCUCCCUUUCUCUGUNN 2686 ACAGAGAAAGGGAGGCUGGNN 37 772-790 CAGCCUCCCUUUCUCUGUCNN 2687 GACAGAGAAAGGGAGGCUGNN 37 773-791 AGCCUCCCUUUCUCUGUCANN 2688 UGACAGAGAAAGGGAGGCUNN 37 774-792 GCCUCCCUUUCUCUGUCAGNN 2689 CUGACAGAGAAAGGGAGGCNN 37 775-793 CCUCCCUUUCUCUGUCAGUNN 2690 ACUGACAGAGAAAGGGAGGNN 37 776-794 CUCCCUUUCUCUGUCAGUGNN 2691 CACUGACAGAGAAAGGGAGNN 37 777-795 UCCCUUUCUCUGUCAGUGGNN 2692 CCACUGACAGAGAAAGGGANN 37 778-796 CCCUUUCUCUGUCAGUGGGNN 2693 CCCACUGACAGAGAAAGGGNN 37 779-797 CCUUUCUCUGUCAGUGGGGNN 2694 CCCCACUGACAGAGAAAGGNN 37 780-798 CUUUCUCUGUCAGUGGGGANN 2695 UCCCCACUGACAGAGAAAGNN 37 781-799 UUUCUCUGUCAGUGGGGACNN 2696 GUCCCCACUGACAGAGAAANN 37 782-800 UUCUCUGUCAGUGGGGACGNN 2697 CGUCCCCACUGACAGAGAANN 37 783-801 UCUCUGUCAGUGGGGACGUNN 2698 ACGUCCCCACUGACAGAGANN 37 784-802 CUCUGUCAGUGGGGACGUCNN 2699 GACGUCCCCACUGACAGAGNN 37 785-803 UCUGUCAGUGGGGACGUCANN 2700 UGACGUCCCCACUGACAGANN 37 786-804 CUGUCAGUGGGGACGUCAUNN 2701 AUGACGUCCCCACUGACAGNN 37 787-805 UGUCAGUGGGGACGUCAUCNN 2702 GAUGACGUCCCCACUGACANN 37 788-806 GUCAGUGGGGACGUCAUCANN 2703 UGAUGACGUCCCCACUGACNN 37 789-807 UCAGUGGGGACGUCAUCAGNN 2704 CUGAUGACGUCCCCACUGANN 37 790-808 CAGUGGGGACGUCAUCAGCNN 2705 GCUGAUGACGUCCCCACUGNN 37 791-809 AGUGGGGACGUCAUCAGCCNN 2706 GGCUGAUGACGUCCCCACUNN 37 792-810 GUGGGGACGUCAUCAGCCANN 2707 UGGCUGAUGACGUCCCCACNN 37 793-811 UGGGGACGUCAUCAGCCAANN 2708 UUGGCUGAUGACGUCCCCANN 37 794-812 GGGGACGUCAUCAGCCAAGNN 2709 CUUGGCUGAUGACGUCCCCNN 37 795-813 GGGACGUCAUCAGCCAAGCNN 2710 GCUUGGCUGAUGACGUCCCNN 37 796-814 GGACGUCAUCAGCCAAGCUNN 2711 AGCUUGGCUGAUGACGUCCNN 37 797-815 GACGUCAUCAGCCAAGCUGNN 2712 CAGCUUGGCUGAUGACGUCNN 37 798-816 ACGUCAUCAGCCAAGCUGGNN 2713 CCAGCUUGGCUGAUGACGUNN 37 799-817 CGUCAUCAGCCAAGCUGGANN 2714 UCCAGCUUGGCUGAUGACGNN 37 800-818 GUCAUCAGCCAAGCUGGAANN 2715 UUCCAGCUUGGCUGAUGACNN 37 801-819 UCAUCAGCCAAGCUGGAAGNN 2716 CUUCCAGCUUGGCUGAUGANN 37 802-820 CAUCAGCCAAGCUGGAAGCNN 2717 GCUUCCAGCUUGGCUGAUGNN 37 803-821 AUCAGCCAAGCUGGAAGCCNN 2718 GGCUUCCAGCUUGGCUGAUNN 37 804-822 UCAGCCAAGCUGGAAGCCANN 2719 UGGCUUCCAGCUUGGCUGANN 37 805-823 CAGCCAAGCUGGAAGCCAUNN 2720 AUGGCUUCCAGCUUGGCUGNN 37 806-824 AGCCAAGCUGGAAGCCAUUNN 2721 AAUGGCUUCCAGCUUGGCUNN 37 807-825 GCCAAGCUGGAAGCCAUUANN 2722 UAAUGGCUUCCAGCUUGGCNN 37 808-826 CCAAGCUGGAAGCCAUUAANN 2723 UUAAUGGCUUCCAGCUUGGNN 37 809-827 CAAGCUGGAAGCCAUUAAUNN 2724 AUUAAUGGCUUCCAGCUUGNN 37 810-828 AAGCUGGAAGCCAUUAAUGNN 2725 CAUUAAUGGCUUCCAGCUUNN 37 811-829 AGCUGGAAGCCAUUAAUGANN 2726 UCAUUAAUGGCUUCCAGCUNN 37 812-830 GCUGGAAGCCAUUAAUGAANN 2727 UUCAUUAAUGGCUUCCAGCNN 37 813-831 CUGGAAGCCAUUAAUGAACNN 2728 GUUCAUUAAUGGCUUCCAGNN 37 814-832 UGGAAGCCAUUAAUGAACUNN 2729 AGUUCAUUAAUGGCUUCCANN 37 815-833 GGAAGCCAUUAAUGAACUANN 2730 UAGUUCAUUAAUGGCUUCCNN 37 816-834 GAAGCCAUUAAUGAACUAANN 2731 UUAGUUCAUUAAUGGCUUCNN 37 817-835 AAGCCAUUAAUGAACUAAUNN 2732 AUUAGUUCAUUAAUGGCUUNN 37 818-836 AGCCAUUAAUGAACUAAUUNN 2733 AAUUAGUUCAUUAAUGGCUNN 37 819-837 GCCAUUAAUGAACUAAUUCNN 2734 GAAUUAGUUCAUUAAUGGCNN 37 820-838 CCAUUAAUGAACUAAUUCGNN 2735 CGAAUUAGUUCAUUAAUGGNN 37 821-839 CAUUAAUGAACUAAUUCGUNN 2736 ACGAAUUAGUUCAUUAAUGNN 37 822-840 AUUAAUGAACUAAUUCGUUNN 2737 AACGAAUUAGUUCAUUAAUNN 37 823-841 UUAAUGAACUAAUUCGUUUNN 2738 AAACGAAUUAGUUCAUUAANN 37 824-842 UAAUGAACUAAUUCGUUUUNN 2739 AAAACGAAUUAGUUCAUUANN 37 825-843 AAUGAACUAAUUCGUUUUGNN 2740 CAAAACGAAUUAGUUCAUUNN 37 826-844 AUGAACUAAUUCGUUUUGANN 2741 UCAAAACGAAUUAGUUCAUNN 38 827-845 UGAACUAAUUCGUUUUGACNN 2742 GUCAAAACGAAUUAGUUCANN 38 828-846 GAACUAAUUCGUUUUGACCNN 2743 GGUCAAAACGAAUUAGUUCNN 38 829-847 AACUAAUUCGUUUUGACCANN 2744 UGGUCAAAACGAAUUAGUUNN 38 830-848 ACUAAUUCGUUUUGACCACNN 2745 GUGGUCAAAACGAAUUAGUNN 38 831-849 CUAAUUCGUUUUGACCACANN 2746 UGUGGUCAAAACGAAUUAGNN 38 832-850 UAAUUCGUUUUGACCACAUNN 2747 AUGUGGUCAAAACGAAUUANN 38 833-851 AAUUCGUUUUGACCACAUANN 2748 UAUGUGGUCAAAACGAAUUNN 38 834-852 AUUCGUUUUGACCACAUAUNN 2749 AUAUGUGGUCAAAACGAAUNN 38 835-853 UUCGUUUUGACCACAUAUANN 2750 UAUAUGUGGUCAAAACGAANN 38 836-854 UCGUUUUGACCACAUAUAUNN 2751 AUAUAUGUGGUCAAAACGANN 38 837-855 CGUUUUGACCACAUAUAUANN 2752 UAUAUAUGUGGUCAAAACGNN 38 838-856 GUUUUGACCACAUAUAUACNN 2753 GUAUAUAUGUGGUCAAAACNN 38 839-857 UUUUGACCACAUAUAUACCNN 2754 GGUAUAUAUGUGGUCAAAANN 38 840-858 UUUGACCACAUAUAUACCANN 2755 UGGUAUAUAUGUGGUCAAANN 38 841-859 UUGACCACAUAUAUACCAANN 2756 UUGGUAUAUAUGUGGUCAANN 38 842-860 UGACCACAUAUAUACCAAGNN 2757 CUUGGUAUAUAUGUGGUCANN 38 843-861 GACCACAUAUAUACCAAGCNN 2758 GCUUGGUAUAUAUGUGGUCNN 38 844-862 ACCACAUAUAUACCAAGCCNN 2759 GGCUUGGUAUAUAUGUGGUNN 38 845-863 CCACAUAUAUACCAAGCCCNN 2760 GGGCUUGGUAUAUAUGUGGNN 38 846-864 CACAUAUAUACCAAGCCCCNN 2761 GGGGCUUGGUAUAUAUGUGNN 38 847-865 ACAUAUAUACCAAGCCCCUNN 2762 AGGGGCUUGGUAUAUAUGUNN 38 867-885 GUCUUAGAGAUACCCUCUGNN 2763 CAGAGGGUAUCUCUAAGACNN 38 868-886 UCUUAGAGAUACCCUCUGANN 2764 UCAGAGGGUAUCUCUAAGANN 38 869-887 CUUAGAGAUACCCUCUGAGNN 2765 CUCAGAGGGUAUCUCUAAGNN 38 870-888 UUAGAGAUACCCUCUGAGANN 2766 UCUCAGAGGGUAUCUCUAANN 38 871-889 UAGAGAUACCCUCUGAGACNN 2767 GUCUCAGAGGGUAUCUCUANN 38 872-890 AGAGAUACCCUCUGAGACANN 2768 UGUCUCAGAGGGUAUCUCUNN 38 873-891 GAGAUACCCUCUGAGACAGNN 2769 CUGUCUCAGAGGGUAUCUCNN 38 874-892 AGAUACCCUCUGAGACAGANN 2770 UCUGUCUCAGAGGGUAUCUNN 38 875-893 GAUACCCUCUGAGACAGAGNN 2771 CUCUGUCUCAGAGGGUAUCNN 38 876-894 AUACCCUCUGAGACAGAGANN 2772 UCUCUGUCUCAGAGGGUAUNN 38 877-895 UACCCUCUGAGACAGAGAGNN 2773 CUCUCUGUCUCAGAGGGUANN 38 878-896 ACCCUCUGAGACAGAGAGCNN 2774 GCUCUCUGUCUCAGAGGGUNN 38 879-897 CCCUCUGAGACAGAGAGCCNN 2775 GGCUCUCUGUCUCAGAGGGNN 38 880-898 CCUCUGAGACAGAGAGCCANN 2776 UGGCUCUCUGUCUCAGAGGNN 38 881-899 CUCUGAGACAGAGAGCCAANN 2777 UUGGCUCUCUGUCUCAGAGNN 38 882-900 UCUGAGACAGAGAGCCAAGNN 2778 CUUGGCUCUCUGUCUCAGANN 38 883-901 CUGAGACAGAGAGCCAAGCNN 2779 GCUUGGCUCUCUGUCUCAGNN 38 884-902 UGAGACAGAGAGCCAAGCUNN 2780 AGCUUGGCUCUCUGUCUCANN 38 885-903 GAGACAGAGAGCCAAGCUANN 2781 UAGCUUGGCUCUCUGUCUCNN 38 886-904 AGACAGAGAGCCAAGCUAANN 2782 UUAGCUUGGCUCUCUGUCUNN 38 887-905 GACAGAGAGCCAAGCUAAUNN 2783 AUUAGCUUGGCUCUCUGUCNN 38 888-906 ACAGAGAGCCAAGCUAAUGNN 2784 CAUUAGCUUGGCUCUCUGUNN 38 889-907 CAGAGAGCCAAGCUAAUGUNN 2785 ACAUUAGCUUGGCUCUCUGNN 38 890-908 AGAGAGCCAAGCUAAUGUGNN 2786 CACAUUAGCUUGGCUCUCUNN 38 891-909 GAGAGCCAAGCUAAUGUGGNN 2787 CCACAUUAGCUUGGCUCUCNN 38 892-910 AGAGCCAAGCUAAUGUGGUNN 2788 ACCACAUUAGCUUGGCUCUNN 38 893-911 GAGCCAAGCUAAUGUGGUANN 2789 UACCACAUUAGCUUGGCUCNN 38 894-912 AGCCAAGCUAAUGUGGUAGNN 2790 CUACCACAUUAGCUUGGCUNN 38 895-913 GCCAAGCUAAUGUGGUAGUNN 2791 ACUACCACAUUAGCUUGGCNN 38 896-914 CCAAGCUAAUGUGGUAGUGNN 2792 CACUACCACAUUAGCUUGGNN 38 897-915 CAAGCUAAUGUGGUAGUGANN 2793 UCACUACCACAUUAGCUUGNN 38 898-916 AAGCUAAUGUGGUAGUGAANN 2794 UUCACUACCACAUUAGCUUNN 38 899-917 AGCUAAUGUGGUAGUGAAANN 2795 UUUCACUACCACAUUAGCUNN 38 900-918 GCUAAUGUGGUAGUGAAAANN 2796 UUUUCACUACCACAUUAGCNN 38 901-919 CUAAUGUGGUAGUGAAAAUNN 2797 AUUUUCACUACCACAUUAGNN 38 902-920 UAAUGUGGUAGUGAAAAUCNN 2798 GAUUUUCACUACCACAUUANN 38 903-921 AAUGUGGUAGUGAAAAUCGNN 2799 CGAUUUUCACUACCACAUUNN 38 904-922 AUGUGGUAGUGAAAAUCGANN 2800 UCGAUUUUCACUACCACAUNN 38 905-923 UGUGGUAGUGAAAAUCGAGNN 2801 CUCGAUUUUCACUACCACANN 38 906-924 GUGGUAGUGAAAAUCGAGGNN 2802 CCUCGAUUUUCACUACCACNN 38 907-925 UGGUAGUGAAAAUCGAGGANN 2803 UCCUCGAUUUUCACUACCANN 38 908-926 GGUAGUGAAAAUCGAGGAANN 2804 UUCCUCGAUUUUCACUACCNN 38 909-927 GUAGUGAAAAUCGAGGAAGNN 2805 CUUCCUCGAUUUUCACUACNN 38 910-928 UAGUGAAAAUCGAGGAAGCNN 2806 GCUUCCUCGAUUUUCACUANN 38 911-929 AGUGAAAAUCGAGGAAGCANN 2807 UGCUUCCUCGAUUUUCACUNN 38 912-930 GUGAAAAUCGAGGAAGCACNN 2808 GUGCUUCCUCGAUUUUCACNN 38 913-931 UGAAAAUCGAGGAAGCACCNN 2809 GGUGCUUCCUCGAUUUUCANN 38 914-932 GAAAAUCGAGGAAGCACCUNN 2810 AGGUGCUUCCUCGAUUUUCNN 38 915-933 AAAAUCGAGGAAGCACCUCNN 2811 GAGGUGCUUCCUCGAUUUUNN 38 916-934 AAAUCGAGGAAGCACCUCUNN 2812 AGAGGUGCUUCCUCGAUUUNN 38 917-935 AAUCGAGGAAGCACCUCUCNN 2813 GAGAGGUGCUUCCUCGAUUNN 38 918-936 AUCGAGGAAGCACCUCUCANN 2814 UGAGAGGUGCUUCCUCGAUNN 38 919-937 UCGAGGAAGCACCUCUCAGNN 2815 CUGAGAGGUGCUUCCUCGANN 38 920-938 CGAGGAAGCACCUCUCAGCNN 2816 GCUGAGAGGUGCUUCCUCGNN 38 921-939 GAGGAAGCACCUCUCAGCCNN 2817 GGCUGAGAGGUGCUUCCUCNN 38 922-940 AGGAAGCACCUCUCAGCCCNN 2818 GGGCUGAGAGGUGCUUCCUNN 38 923-941 GGAAGCACCUCUCAGCCCCNN 2819 GGGGCUGAGAGGUGCUUCCNN 38 924-942 GAAGCACCUCUCAGCCCCUNN 2820 AGGGGCUGAGAGGUGCUUCNN 38 925-943 AAGCACCUCUCAGCCCCUCNN 2821 GAGGGGCUGAGAGGUGCUUNN 38 926-944 AGCACCUCUCAGCCCCUCANN 2822 UGAGGGGCUGAGAGGUGCUNN 38 927-945 GCACCUCUCAGCCCCUCAGNN 2823 CUGAGGGGCUGAGAGGUGCNN 38 928-946 CACCUCUCAGCCCCUCAGANN 2824 UCUGAGGGGCUGAGAGGUGNN 38 929-947 ACCUCUCAGCCCCUCAGAGNN 2825 CUCUGAGGGGCUGAGAGGUNN 38 930-948 CCUCUCAGCCCCUCAGAGANN 2826 UCUCUGAGGGGCUGAGAGGNN 38 931-949 CUCUCAGCCCCUCAGAGAANN 2827 UUCUCUGAGGGGCUGAGAGNN 38 932-950 UCUCAGCCCCUCAGAGAAUNN 2828 AUUCUCUGAGGGGCUGAGANN 38 933-951 CUCAGCCCCUCAGAGAAUGNN 2829 CAUUCUCUGAGGGGCUGAGNN 38 934-952 UCAGCCCCUCAGAGAAUGANN 2830 UCAUUCUCUGAGGGGCUGANN 38 935-953 CAGCCCCUCAGAGAAUGAUNN 2831 AUCAUUCUCUGAGGGGCUGNN 38 936-954 AGCCCCUCAGAGAAUGAUCNN 2832 GAUCAUUCUCUGAGGGGCUNN 38 937-955 GCCCCUCAGAGAAUGAUCANN 2833 UGAUCAUUCUCUGAGGGGCNN 38 938-956 CCCCUCAGAGAAUGAUCACNN 2834 GUGAUCAUUCUCUGAGGGGNN 38 939-957 CCCUCAGAGAAUGAUCACCNN 2835 GGUGAUCAUUCUCUGAGGGNN 38 940-958 CCUCAGAGAAUGAUCACCCNN 2836 GGGUGAUCAUUCUCUGAGGNN 38 941-959 CUCAGAGAAUGAUCACCCUNN 2837 AGGGUGAUCAUUCUCUGAGNN 38 942-960 UCAGAGAAUGAUCACCCUGNN 2838 CAGGGUGAUCAUUCUCUGANN 38 943-961 CAGAGAAUGAUCACCCUGANN 2839 UCAGGGUGAUCAUUCUCUGNN 38 944-962 AGAGAAUGAUCACCCUGAANN 2840 UUCAGGGUGAUCAUUCUCUNN 38 945-963 GAGAAUGAUCACCCUGAAUNN 2841 AUUCAGGGUGAUCAUUCUCNN 39 946-964 AGAAUGAUCACCCUGAAUUNN 2842 AAUUCAGGGUGAUCAUUCUNN 39 947-965 GAAUGAUCACCCUGAAUUCNN 2843 GAAUUCAGGGUGAUCAUUCNN 39 948-966 AAUGAUCACCCUGAAUUCANN 2844 UGAAUUCAGGGUGAUCAUUNN 39 949-967 AUGAUCACCCUGAAUUCAUNN 2845 AUGAAUUCAGGGUGAUCAUNN 39 950-968 UGAUCACCCUGAAUUCAUUNN 2846 AAUGAAUUCAGGGUGAUCANN 39 951-969 GAUCACCCUGAAUUCAUUGNN 2847 CAAUGAAUUCAGGGUGAUCNN 39 952-970 AUCACCCUGAAUUCAUUGUNN 2848 ACAAUGAAUUCAGGGUGAUNN 39 953-971 UCACCCUGAAUUCAUUGUCNN 2849 GACAAUGAAUUCAGGGUGANN 39 954-972 CACCCUGAAUUCAUUGUCUNN 2850 AGACAAUGAAUUCAGGGUGNN 39 955-973 ACCCUGAAUUCAUUGUCUCNN 2851 GAGACAAUGAAUUCAGGGUNN 39 956-974 CCCUGAAUUCAUUGUCUCANN 2852 UGAGACAAUGAAUUCAGGGNN 39 957-975 CCUGAAUUCAUUGUCUCAGNN 2853 CUGAGACAAUGAAUUCAGGNN 39 958-976 CUGAAUUCAUUGUCUCAGUNN 2854 ACUGAGACAAUGAAUUCAGNN 39 959-977 UGAAUUCAUUGUCUCAGUGNN 2855 CACUGAGACAAUGAAUUCANN 39 960-978 GAAUUCAUUGUCUCAGUGANN 2856 UCACUGAGACAAUGAAUUCNN 39 961-979 AAUUCAUUGUCUCAGUGAANN 2857 UUCACUGAGACAAUGAAUUNN 39 962-980 AUUCAUUGUCUCAGUGAAGNN 2858 CUUCACUGAGACAAUGAAUNN 39 963-981 UUCAUUGUCUCAGUGAAGGNN 2859 CCUUCACUGAGACAAUGAANN 39 964-982 UCAUUGUCUCAGUGAAGGANN 2860 UCCUUCACUGAGACAAUGANN 39 965-983 CAUUGUCUCAGUGAAGGAANN 2861 UUCCUUCACUGAGACAAUGNN 39 966-984 AUUGUCUCAGUGAAGGAAGNN 2862 CUUCCUUCACUGAGACAAUNN 39 967-985 UUGUCUCAGUGAAGGAAGANN 2863 UCUUCCUUCACUGAGACAANN 39 968-986 UGUCUCAGUGAAGGAAGAANN 2864 UUCUUCCUUCACUGAGACANN 39 969-987 GUCUCAGUGAAGGAAGAACNN 2865 GUUCUUCCUUCACUGAGACNN 39 970-988 UCUCAGUGAAGGAAGAACCNN 2866 GGUUCUUCCUUCACUGAGANN 39 971-989 CUCAGUGAAGGAAGAACCUNN 2867 AGGUUCUUCCUUCACUGAGNN 39 972-990 UCAGUGAAGGAAGAACCUGNN 2868 CAGGUUCUUCCUUCACUGANN 39 973-991 CAGUGAAGGAAGAACCUGUNN 2869 ACAGGUUCUUCCUUCACUGNN 39 974-992 AGUGAAGGAAGAACCUGUANN 2870 UACAGGUUCUUCCUUCACUNN 39 975-993 GUGAAGGAAGAACCUGUAGNN 2871 CUACAGGUUCUUCCUUCACNN 39 976-994 UGAAGGAAGAACCUGUAGANN 2872 UCUACAGGUUCUUCCUUCANN 39 977-995 GAAGGAAGAACCUGUAGAANN 2873 UUCUACAGGUUCUUCCUUCNN 39 978-996 AAGGAAGAACCUGUAGAAGNN 2874 CUUCUACAGGUUCUUCCUUNN 39 979-997 AGGAAGAACCUGUAGAAGANN 2875 UCUUCUACAGGUUCUUCCUNN 39 980-998 GGAAGAACCUGUAGAAGAUNN 2876 AUCUUCUACAGGUUCUUCCNN 39 981-999 GAAGAACCUGUAGAAGAUGNN 2877 CAUCUUCUACAGGUUCUUCNN 39 982-1000 AAGAACCUGUAGAAGAUGANN 2878 UCAUCUUCUACAGGUUCUUNN 39 983-1001 AGAACCUGUAGAAGAUGACNN 2879 GUCAUCUUCUACAGGUUCUNN 39 984-1002 GAACCUGUAGAAGAUGACCNN 2880 GGUCAUCUUCUACAGGUUCNN 39 985-1003 AACCUGUAGAAGAUGACCUNN 2881 AGGUCAUCUUCUACAGGUUNN 39 986-1004 ACCUGUAGAAGAUGACCUCNN 2882 GAGGUCAUCUUCUACAGGUNN 39 *Target refers location of target sequence in NM_005080 (human XBP-1 mRNA). Sense and antisense sequences are described with optional dinucleotide (NN) overhangs.

TABLE 13 Sequences of dsRNA targeting both mouse and rhesus monkeyXBP-1. SEQ ID SEQ ID *Target sense (5′-3′) NO antisense (5′-3′) NO 369-387 AGAAAACUCACGGCCUUGUNN 3942 ACAAGGCCGUGAGUUUUCUNN 4042 237-255 AACUGAAAAACAGAGUAGCNN 3943 GCUACUCUGUUUUUCAGUUNN 4043 491-509 GGGUCUGCUGAGUCCGCAGNN 3944 CUGCGGACUCAGCAGACCCNN 4044 917-935 AUCACCCUGAAUUCAUUGUNN 3945 ACAAUGAAUUCAGGGUGAUNN 4045 923-941 CUGAAUUCAUUGUCUCAGUNN 3946 ACUGAGACAAUGAAUUCAGNN 4046 702-720 CCCAGAGGUCUACCCAGAANN 3947 UUCUGGGUAGACCUCUGGGNN 4047 926-944 AAUUCAUUGUCUCAGUGAANN 3948 UUCACUGAGACAAUGAAUUNN 4048 391-409 UGAGAACCAGGAGUUAAGANN 3949 UCUUAACUCCUGGUUCUCANN 4049 775-793 AAGCUGGAAGCCAUUAAUGNN 3950 CAUUAAUGGCUUCCAGCUUNN 4050 1150-1168 CCCCAGCUGAUUAGUGUCUNN 3951 AGACACUAAUCAGCUGGGGNN 4051 776-794 AGCUGGAAGCCAUUAAUGANN 3952 UCAUUAAUGGCUUCCAGCUNN 4052 921-939 CCCUGAAUUCAUUGUCUCANN 3953 UGAGACAAUGAAUUCAGGGNN 4053 777-795 GCUGGAAGCCAUUAAUGAANN 3954 UUCAUUAAUGGCUUCCAGCNN 4054 539-557 GUGCAGGCCCAGUUGUCACNN 3955 GUGACAACUGGGCCUGCACNN 4055 731-749 CCUUACCAGCCUCCCUUUCNN 3956 GAAAGGGAGGCUGGUAAGGNN 4056 924-942 UGAAUUCAUUGUCUCAGUGNN 3957 CACUGAGACAAUGAAUUCANN 4057 1151-1169 CCCAGCUGAUUAGUGUCUANN 3958 UAGACACUAAUCAGCUGGGNN 4058 1152-1170 CCAGCUGAUUAGUGUCUAANN 3959 UUAGACACUAAUCAGCUGGNN 4059 1718-1736 ACUAUGUAAAUGCUUGAUGNN 3960 CAUCAAGCAUUUACAUAGUNN 4060 368-386 GAGAAAACUCACGGCCUUGNN 3961 CAAGGCCGUGAGUUUUCUCNN 4061 489-507 CCGGGUCUGCUGAGUCCGCNN 3962 GCGGACUCAGCAGACCCGGNN 4062 238-256 ACUGAAAAACAGAGUAGCANN 3963 UGCUACUCUGUUUUUCAGUNN 4063 240-258 UGAAAAACAGAGUAGCAGCNN 3964 GCUGCUACUCUGUUUUUCANN 4064 390-408 UUGAGAACCAGGAGUUAAGNN 3965 CUUAACUCCUGGUUCUCAANN 4065 487-505 GGCCGGGUCUGCUGAGUCCNN 3966 GGACUCAGCAGACCCGGCCNN 4066 741-759 CUCCCUUUCUCUGUCAGUGNN 3967 CACUGACAGAGAAAGGGAGNN 4067 918-936 UCACCCUGAAUUCAUUGUCNN 3968 GACAAUGAAUUCAGGGUGANN 4068 919-937 CACCCUGAAUUCAUUGUCUNN 3969 AGACAAUGAAUUCAGGGUGNN 4069 1130-1148 CUUUUGCCAAUGAACUUUUNN 3970 AAAAGUUCAUUGGCAAAAGNN 4070 1712-1730 AAAUUUACUAUGUAAAUGCNN 3971 GCAUUUACAUAGUAAAUUUNN 4071 1714-1732 AUUUACUAUGUAAAUGCUUNN 3972 AAGCAUUUACAUAGUAAAUNN 4072 1717-1735 UACUAUGUAAAUGCUUGAUNN 3973 AUCAAGCAUUUACAUAGUANN 4073 1719-1737 CUAUGUAAAUGCUUGAUGGNN 3974 CCAUCAAGCAUUUACAUAGNN 4074 1775-1793 CCAUUUAUUUAAAACUACCNN 3975 GGUAGUUUUAAAUAAAUGGNN 4075 1776-1794 CAUUUAUUUAAAACUACCCNN 3976 GGGUAGUUUUAAAUAAAUGNN 4076 239-257 CUGAAAAACAGAGUAGCAGNN 3977 CUGCUACUCUGUUUUUCAGNN 4077 347-365 CUAGAAAAUCAGCUUUUACNN 3978 GUAAAAGCUGAUUUUCUAGNN 4078 348-366 UAGAAAAUCAGCUUUUACGNN 3979 CGUAAAAGCUGAUUUUCUANN 4079 485-503 GUGGCCGGGUCUGCUGAGUNN 3980 ACUCAGCAGACCCGGCCACNN 4080 486-504 UGGCCGGGUCUGCUGAGUCNN 3981 GACUCAGCAGACCCGGCCANN 4081 488-506 GCCGGGUCUGCUGAGUCCGNN 3982 CGGACUCAGCAGACCCGGCNN 4082 540-558 UGCAGGCCCAGUUGUCACCNN 3983 GGUGACAACUGGGCCUGCANN 4083 703-721 CCAGAGGUCUACCCAGAAGNN 3984 CUUCUGGGUAGACCUCUGGNN 4084 705-723 AGAGGUCUACCCAGAAGGANN 3985 UCCUUCUGGGUAGACCUCUNN 4085 730-748 UCCUUACCAGCCUCCCUUUNN 3986 AAAGGGAGGCUGGUAAGGANN 4086 742-760 UCCCUUUCUCUGUCAGUGGNN 3987 CCACUGACAGAGAAAGGGANN 4087 744-762 CCUUUCUCUGUCAGUGGGGNN 3988 CCCCACUGACAGAGAAAGGNN 4088 767-785 CAUCAGCCAAGCUGGAAGCNN 3989 GCUUCCAGCUUGGCUGAUGNN 4089 771-789 AGCCAAGCUGGAAGCCAUUNN 3990 AAUGGCUUCCAGCUUGGCUNN 4090 916-934 GAUCACCCUGAAUUCAUUGNN 3991 CAAUGAAUUCAGGGUGAUCNN 4091 920-938 ACCCUGAAUUCAUUGUCUCNN 3992 GAGACAAUGAAUUCAGGGUNN 4092 922-940 CCUGAAUUCAUUGUCUCAGNN 3993 CUGAGACAAUGAAUUCAGGNN 4093 925-943 GAAUUCAUUGUCUCAGUGANN 3994 UCACUGAGACAAUGAAUUCNN 4094 1720-1738 UAUGUAAAUGCUUGAUGGANN 3995 UCCAUCAAGCAUUUACAUANN 4095 232-250 GAGGAAACUGAAAAACAGANN 3996 UCUGUUUUUCAGUUUCCUCNN 4096 236-254 AAACUGAAAAACAGAGUAGNN 3997 CUACUCUGUUUUUCAGUUUNN 4097 728-746 GUUCCUUACCAGCCUCCCUNN 3998 AGGGAGGCUGGUAAGGAACNN 4098 729-747 UUCCUUACCAGCCUCCCUUNN 3999 AAGGGAGGCUGGUAAGGAANN 4099 745-763 CUUUCUCUGUCAGUGGGGANN 4000 UCCCCACUGACAGAGAAAGNN 4100 766-784 UCAUCAGCCAAGCUGGAAGNN 4001 CUUCCAGCUUGGCUGAUGANN 4101 927-945 AUUCAUUGUCUCAGUGAAGNN 4002 CUUCACUGAGACAAUGAAUNN 4102 234-252 GGAAACUGAAAAACAGAGUNN 4003 ACUCUGUUUUUCAGUUUCCNN 4103 235-253 GAAACUGAAAAACAGAGUANN 4004 UACUCUGUUUUUCAGUUUCNN 4104 346-364 GCUAGAAAAUCAGCUUUUANN 4005 UAAAAGCUGAUUUUCUAGCNN 4105 490-508 CGGGUCUGCUGAGUCCGCANN 4006 UGCGGACUCAGCAGACCCGNN 4106 700-718 CUCCCAGAGGUCUACCCAGNN 4007 CUGGGUAGACCUCUGGGAGNN 4107 1715-1733 UUUACUAUGUAAAUGCUUGNN 4008 CAAGCAUUUACAUAGUAAANN 4108 734-752 UACCAGCCUCCCUUUCUCUNN 4009 AGAGAAAGGGAGGCUGGUANN 4109 773-791 CCAAGCUGGAAGCCAUUAANN 4010 UUAAUGGCUUCCAGCUUGGNN 4110 778-796 CUGGAAGCCAUUAAUGAACNN 4011 GUUCAUUAAUGGCUUCCAGNN 4111 779-797 UGGAAGCCAUUAAUGAACUNN 4012 AGUUCAUUAAUGGCUUCCANN 4112 1774-1792 UCCAUUUAUUUAAAACUACNN 4013 GUAGUUUUAAAUAAAUGGANN 4113 704-722 CAGAGGUCUACCCAGAAGGNN 4014 CCUUCUGGGUAGACCUCUGNN 4114 1716-1734 UUACUAUGUAAAUGCUUGANN 4015 UCAAGCAUUUACAUAGUAANN 4115 1713-1731 AAUUUACUAUGUAAAUGCUNN 4016 AGCAUUUACAUAGUAAAUUNN 4116 768-786 AUCAGCCAAGCUGGAAGCCNN 4017 GGCUUCCAGCUUGGCUGAUNN 4117 1129-1147 ACUUUUGCCAAUGAACUUUNN 4018 AAAGUUCAUUGGCAAAAGUNN 4118 389-407 GUUGAGAACCAGGAGUUAANN 4019 UUAACUCCUGGUUCUCAACNN 4119 701-719 UCCCAGAGGUCUACCCAGANN 4020 UCUGGGUAGACCUCUGGGANN 4120 706-724 GAGGUCUACCCAGAAGGACNN 4021 GUCCUUCUGGGUAGACCUCNN 4121 707-725 AGGUCUACCCAGAAGGACCNN 4022 GGUCCUUCUGGGUAGACCUNN 4122 727-745 AGUUCCUUACCAGCCUCCCNN 4023 GGGAGGCUGGUAAGGAACUNN 4123 733-751 UUACCAGCCUCCCUUUCUCNN 4024 GAGAAAGGGAGGCUGGUAANN 4124 736-754 CCAGCCUCCCUUUCUCUGUNN 4025 ACAGAGAAAGGGAGGCUGGNN 4125 738-756 AGCCUCCCUUUCUCUGUCANN 4026 UGACAGAGAAAGGGAGGCUNN 4126 743-761 CCCUUUCUCUGUCAGUGGGNN 4027 CCCACUGACAGAGAAAGGGNN 4127 769-787 UCAGCCAAGCUGGAAGCCANN 4028 UGGCUUCCAGCUUGGCUGANN 4128 772-790 GCCAAGCUGGAAGCCAUUANN 4029 UAAUGGCUUCCAGCUUGGCNN 4129 774-792 CAAGCUGGAAGCCAUUAAUNN 4030 AUUAAUGGCUUCCAGCUUGNN 4130 231-249 GGAGGAAACUGAAAAACAGNN 4031 CUGUUUUUCAGUUUCCUCCNN 4131 233-251 AGGAAACUGAAAAACAGAGNN 4032 CUCUGUUUUUCAGUUUCCUNN 4132 735-753 ACCAGCCUCCCUUUCUCUGNN 4033 CAGAGAAAGGGAGGCUGGUNN 4133 737-755 CAGCCUCCCUUUCUCUGUCNN 4034 GACAGAGAAAGGGAGGCUGNN 4134 739-757 GCCUCCCUUUCUCUGUCAGNN 4035 CUGACAGAGAAAGGGAGGCNN 4135 740-758 CCUCCCUUUCUCUGUCAGUNN 4036 ACUGACAGAGAAAGGGAGGNN 4136 746-764 UUUCUCUGUCAGUGGGGACNN 4037 GUCCCCACUGACAGAGAAANN 4137 770-788 CAGCCAAGCUGGAAGCCAUNN 4038 AUGGCUUCCAGCUUGGCUGNN 4138 26-44 GCUAUGGUGGUGGUGGCAGNN 4039 CUGCCACCACCACCAUAGCNN 4139 27-45 CUAUGGUGGUGGUGGCAGCNN 4040 GCUGCCACCACCACCAUAGNN 4140 732-750 CUUACCAGCCUCCCUUUCUNN 4041 AGAAAGGGAGGCUGGUAAGNN 4141 *Target refers location of target sequence in NM_013842 (Mus musculis XPB1 mRNA). Sense and antisense sequences are described with optional dinucleotide (NN) overhangs. 

1. A dual targeting siRNA agent comprising a first dsRNA targeting a PCSK9 gene and a second dsRNA targeting a second gene, wherein the first dsRNA and the second dsRNA are linked with a covalent linker wherein the first dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 1, 2, or 4-8, or comprises an antisense strand of one of Tables 1, 2, or 4-8, or comprises a sense strand and an antisense strand of one of Tables 1, 2, or 4-8 and the second dsRNA comprises at least 15 contiguous nucleotides of an antisense strand of one of Tables 3 or 9-13, or comprises an antisense strand of one of Tables 3 or 9-13, or comprises a sense strand and an antisense strand of one of Tables 3 or 9-13.
 2. (canceled)
 3. The dual targeting siRNA agent of claim 1, wherein the second gene is selected from the group consisting of XBP-1, PCSK9, PCSK5, ApoC3, SCAP, and MIG12.
 4. The dual targeting siRNA agent of claim 1, wherein the second gene is XBP-1.
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. The dual targeting siRNA agent of claim 1, wherein the first and second dsRNA comprises at least one modified nucleotide.
 10. The dual targeting siRNA agent of claim 9, wherein the modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
 11. The dual targeting siRNA agent of claim 9, wherein the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
 12. The dual targeting siRNA agent of claim 1, wherein each strand of each dsRNA is 19-23 bases in length.
 13. The dual targeting siRNA agent of claim 1, wherein the first and second dsRNAs are linked with a disulfide linker.
 14. The dual targeting siRNA agent of claim 1, wherein the covalent linker links the sense strand of the first dsRNA to the sense strand of the second dsRNA.
 15. The dual targeting siRNA agent of claim 1, wherein the covalent linker links the antisense strand of the first dsRNA to the antisense strand of the second dsRNA.
 16. The dual targeting siRNA agent of claim 1, further comprising a ligand.
 17. The dual targeting siRNA agent of claim 1, wherein administration of the dual targeting siRNA agent to a cell inhibits expression of the PCSK9 gene and the second gene at a level equivalent to inhibition of expression of both genes obtained by the administration of each siRNA individually.
 18. The dual targeting siRNA agent of claim 1, wherein administration of the dual targeting siRNA agent to a subject results in a greater reduction of total serum cholesterol that that obtained by administration of each siRNA individually.
 19. A pharmaceutical composition comprising the dual targeting siRNA agent of claim 1 and a pharmaceutical carrier.
 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical carrier is a lipid formulation.
 21. (canceled)
 22. The pharmaceutical composition of claim 19, wherein the pharmaceutical carrier is a lipid formulation described in Table A.
 23. (canceled)
 24. A method of inhibiting expression of the PCSK9 gene and a second gene in a cell, the method comprising (a) introducing into the cell the dual targeting siRNA agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PCSK9 gene and the second gene, thereby inhibiting expression of the PCSK9 gene and the second gene in the cell.
 25. (canceled)
 26. (canceled)
 27. A method of reducing total serum cholesterol in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim
 1. 28. An isolated cell comprising the dual targeting siRNA agent of claim
 1. 29. (canceled)
 30. (canceled) 